Artificial antibody polypeptides

ABSTRACT

A fibronectin type III (Fn3) polypeptide monobody, a nucleic acid molecule encoding said monobody, and a variegated nucleic acid library encoding said monobody, are provided by the invention. Also provided are methods of preparing a Fn3 polypeptide monobody, and kits to perform said methods. Further provided is a method of identifying the amino acid sequence of a polypeptide molecule capable of binding to a specific binding partner (SBP) so as to form a polypeptide:SSP complex, and a method of identifying the amino acid sequence of a polypeptide molecule capable of catalyzing a chemical reaction with a catalyzed rate constant, k cat , and an uncatalyzed rate constant, k uncat , such that the ratio of k cat /k uncat , is greater than 10.

RELATED APPLICATIONS

This application is a continuation under 37 C.F.R 1.53(b) of U.S. Ser.No. 09/096,749, filed Jun. 12, 1998, which claims priority under 35U.S.C. 119(e) to provisional application U.S. Ser. No. 60/049,410, filedJun. 12, 1997, which applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the field of the productionand selection of binding and catalytic polypeptides by the methods ofmolecular biology, using both combinatorial chemistry and recombinantDNA. The invention specifically relates to the generation of bothnucleic acid and polypeptide libraries derived therefrom encoding themolecular scaffolding of Fibronectin Type m (Fn3) modified in one ormore of its loop regions. The invention also relates to the “artificialmini-antibodies” or “monobodies,” i.e., the polypeptides comprising anFn3 scaffold onto which loop regions capable of binding to a variety ofdifferent molecular structures (such as antibody binding sites) havebeen grafted.

BACKGROUND OF THE INVENTION

Antibody Structure

A standard antibody (Ab) is a tetrameric structure consisting of twoidentical immunoglobulin (Ig) heavy chains and two identical lightchains. The heavy and light chains of an Ab consist of differentdomains. Each light chain has one variable domain (VL) and one constantdomain (CL), while each heavy chain has one variable domain (VH) andthree or four constant domains (CH) (Alzari et al., 1988). Each domain,consisting of ˜110 amino acid residues, is folded into a characteristicβ-sandwich structure formed from two β-sheets packed against each other,the immunoglobulin fold. The VH and VL domains each have threecomplementarity determining regions (CDR1-3) that are loops, or turns,connecting β-strands at one end of the domains (FIG. 1: A, C). Thevariable regions of both the light and heavy chains generally contributeto antigen specificity, although the contribution of the individualchains to specificity is not always equal. Antibody molecules haveevolved to bind to a large number of molecules by using six randomizedloops (CDRs). However, the size of the antibodies and the complexity ofsix loops represents a major design hurdle if the end result is to be arelatively small peptide ligand.

Antibody Substructures

Functional substructures of Abs can be prepared by proteolysis and byrecombinant methods. They include the Fab fragment, which comprises theVH-CH1 domains of the heavy chain and the VL-CL1 domains of the lightchain joined by a single interchain disulfide bond, and the Fv fragment,which comprises only the VH and VL domains. In some cases, a single VHdomain retains significant affinity (Ward et al., 1989). It has alsobeen shown that a certain monomeric κ light chain will specifically bindto its cognate antigen. (L. Masat et al., 1994). Separated light orheavy chains have sometimes been found to retain some antigen-bindingactivity (Ward et al., 1989). These antibody fragments are not suitablefor structural analysis using NMR spectroscopy due to their size, lowsolubility or low conformational stability.

Another functional substructure is a single chain Fv (scFv), comprisedof the variable regions of the immunoglobulin heavy and light chain,covalently connected by a peptide linker (S-z Hu et al., 1996). Thesesmall (M, 25,000) proteins generally retain specificity and affinity forantigen in a single polypeptide and can provide a convenient buildingblock for larger, antigen-specific molecules. Several groups havereported biodistribution studies in xenografted athymic mice using scFvreactive against a variety of tumor antigens, in which specific tumorlocalization has been observed. However, the short persistence of scFvsin the circulation limits the exposure of tumor cells to the scFvs,placing limits on the level of uptake. As a result, tumor uptake byscFvs in animal studies has generally been only 1-5% ID/g as opposed tointact antibodies that can localize in tumors ad 30-40% ID/g and havereached levels as high as 60-70% ID/g.

A small protein scaffold called a “minibody” was designed using a partof the Ig VH domain as the template (Pessi et al., 1993). Minibodieswith high affinity (dissociation constant (K_(d)) ˜10⁻⁷ M) tointerleukin-6 were identified by randomizing loops corresponding to CDR1and CDR2 of VH and then selecting mutants using the phage display method(Martin et al., 1994). These experiments demonstrated that the essenceof the Ab function could be transferred to a smaller system. However,the minibody had inherited the limited solubility of the VH domain(Bianchi et al., 1994).

It has been reported that camels (Camelus dromedarius) often lackvariable light chain domains when IgG-like material from their serum isanalyzed, suggesting that sufficient antibody specificity and affinitycan be derived form VH domains (three CDR loops) alone. Davies andRiechmann recently demonstrated that “camelized” VH domains with highaffinity (K_(d) ˜10⁻⁷ M) and high specificity can be generated byrandomizing only the CDR3. To improve the solubility and suppressnonspecific binding, three mutations were introduced to the frameworkregion (Davies & Riechmann, 1995). It has not been definitively shown,however, that camelization can be used, in general, to improve thesolubility and stability of VHs.

An alternative to the “minibody” is the “diabody.” Diabodies are smallbivalent and bispecific antibody fragments, i.e., they have twoantigen-binding sites. The fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) on thesame polypeptide chain (V_(H)-V_(L)). Diabodies are similar in size toan Fab fragment. By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. These dimeric antibody fragments, or “diabodies,”are bivalent and bispecific. P. Holliger et al., PNAS 90:6444-6448(1993).

Since the development of the monoclonal antibody technology, a largenumber of 3D structures of Ab fragments in the complexed and/or freestates have been solved by X-ray crystallography (Webster et al., 1994;Wilson & Stanfield, 1994). Analysis of Ab structures has revealed thatfive out of the six CDRs have limited numbers of peptide backboneconformations, thereby permitting one to predict the backboneconformation of CDRs using the so-called canonical structures (Lesk &Tramontano, 1992; Rees et al., 1994). The analysis also has revealedthat the CDR3 of the VH domain (VH-CDR3) usually has the largest contactsurface and that its conformation is too diverse for canonicalstructures to be defined; VH-CDR3 is also known to have a largevariation in length (Wu et al., 1993). Therefore, the structures ofcrucial regions of the Ab-antigen interface still need to beexperimentally determined.

Comparison of crystal structures between the free and complexed stateshas revealed several types of conformational rearrangements. Theyinclude side-chain rearrangements, segmental movements, largerearrangements of VH-CDR3 and changes in the relative position of the VHand VL domains (Wilson & Stanfield, 1993). In the free state, CDRs, inparticular those which undergo large conformational changes uponbinding, are expected to be flexible. Since X-ray crystallography is notsuited for characterizing flexible parts of molecules, structuralstudies in the solution state have not been possible to provide dynamicpictures of the conformation of antigen-binding sites.

Mimicking the Antibody-Binding Site

CDR peptides and organic CDR mimetics have been made (Dougall et al.,1994). CDR peptides are short, typically cyclic, peptides whichcorrespond to the amino acid sequences of CDR loops of antibodies. CDRloops are responsible for antibody-antigen interactions. Organic CDRmimetics are peptides corresponding to CDR loops which are attached to ascaffold, e.g., a small organic compound.

CDR peptides and organic CDR mimetics have been shown to retain somebinding affinity (Smyth & von Itzstein, 1994). However, as expected,they are too small and too flexible to maintain full affinity andspecificity. Mouse CDRs have been grafted onto the human Ig frameworkwithout the loss of affinity (Jones et al., 1986; Riechmann et al.,1988), though this “humanization” does not solve the above-mentionedproblems specific to solution studies.

Mimicking Natural Selection Processes of Abs

In the immune system, specific Abs are selected and amplified from alarge library (affinity maturation). The processes can be reproduced invitro using combinatorial library technologies. The successful displayof Ab fragments on the surface of bacteriophage has made it possible togenerate and screen a vast number of CDR mutations (McCafferty et al.,1990; Barbas et al., 1991; Winter et al., 1994). An increasing number ofFabs and Fvs (and their derivatives) is produced by this technique,providing a rich source for structural studies. The combinatorialtechnique can be combined with Ab mimics.

A number of protein domains that could potentially serve as proteinscaffolds have been expressed as fusions with phage capsid proteins.Review in Clackson & Wells, Trends Biotechnol. 12:173-184 (1994).Indeed, several of these protein domains have already been used asscaffolds for displaying random peptide sequences, including bovinepancreatic trypsin inhibitor (Roberts et al., PNAS 89:2429-2433 (1992)),human growth hormone (Lowman et al., Biochemistry 30:10832-10838(1991)), Venturini et al., Protein Peptide Letters 1:70-75 (1994)), andthe IgG binding domain of Streptococcus (O'Neil et al., Techniques inProtein Chemistry V (Crabb, L,. ed.) pp. 517-524, Academic Press, SanDiego (1994)). These scaffolds have displayed a single randomized loopor region.

Researchers have used the small 74 amino acid α-amylase inhibitorTendamistat as a presentation scaffold on the filamentous phage M13(McConnell and Hoess, 1995). Tendamistat is a β-sheet protein fromStreptomyces tendae. It has a number of features that make it anattractive scaffold for peptides, including its small size, stability,and the availability of high resolution NMR and X-ray structural data.Tendamistat's overall topology is similar to that of an immunoglobulindomain, with two β-sheets connected by a series of loops. In contrast toimmunoglobulin domains, the β-sheets of Tendamistat are held togetherwith two rather than one disulfide bond, accounting for the considerablestability of the protein. By analogy with the CDR loops found inimmunoglobulins, the loops the Tendamistat may serve a similar functionand can be easily randomized by in vitro mutagenesis.

Tendamistat, however, is derived from Streptomyces tendae. Thus, whileTendamistat may be antigenic in humans, its small size may reduce orinhibit its antigenicity. Also, Tendamistat's stability is uncertain.Further, the stability that is reported for Tendamistat is attributed tothe presence of two disulfide bonds. Disulfide bonds, however, are asignificant disadvantage to such molecules in that they can be brokenunder reducing conditions and must be properly formed in order to have auseful protein structure. Further, the size of the loops in Tendamistatare relatively small, thus limiting the size of the inserts that can beaccommodated in the scaffold. Moreover, it is well known that formingcorrect disulfide bonds in newly synthesized peptides is notstraightforward. When a protein is expressed in the cytoplasmic space ofE. coli, the most common host bacterium for protein overexpression,disulfide bonds are usually not formed, potentially making it difficultto prepare large quantities of engineered molecules.

Thus, there is an on-going need for small, single-chain artificialantibodies for a variety of therapeutic, diagnostic and catalyticapplications.

SUMMARY OF THE INVENTION

The invention provides a fibronectin type III (Fn3) polypeptide monobodycomprising a plurality of Fn3 β-strand domain sequences that are linkedto a plurality of loop region sequences. One or more of the monobodyloop region sequences of the Fn3 polypeptide vary by deletion, insertionor replacement of at least two amino acids from the corresponding loopregion sequences in wild-type Fn3. The β-strand domains of the monobodyhave at least about 50% total amino acid sequence homology to thecorresponding amino acid sequence of wild-type Fn3's β-strand domainsequences. Preferably, one or more of the loop regions of the monobodycomprise amino acid residues:

i) from 15 to 16 inclusive in an AB loop;

ii) from 22 to 30 inclusive in a BC loop;

iii) from 39 to 45 inclusive in a CD loop;

iv) from 51 to 55 inclusive in a DE loop;

v) from 60 to 66 inclusive in an EF loop; and

vi) from 76 to 87 inclusive in an FG loop.

The invention also provides a nucleic acid molecule encoding a Fn3polypeptide monobody of the invention, as well as an expression vectorcomprising said nucleic acid molecule and a host cell comprising saidvector.

The invention further provides a method of preparing a Fn3 polypeptidemonobody. The method comprises providing a DNA sequence encoding aplurality of Fn3 β-strand domain sequences that are linked to aplurality of loop region sequences, wherein at least one loop region ofsaid sequence contains a unique restriction enzyme site. The DNAsequence is cleaved at the unique restriction site. Then a preselectedDNA segment is inserted into the restriction site. The preselected DNAsegment encodes a peptide capable of binding to a specific bindingpartner (SBP) or a transition state analog compound (TSAC). Theinsertion of the preselected DNA segment into the DNA sequence yields aDNA molecule which encodes a polypeptide monobody having an insertion.The DNA molecule is then expressed so as to yield the polypeptidemonobody.

Also provided is a method of preparing a Fn3 polypeptide monobody, whichmethod comprises providing a replicatable DNA sequence encoding aplurality of Fn3 β-strand domain sequences that are linked to aplurality of loop region sequences, wherein the nucleotide sequence ofat least one loop region is known. Polymerase chain reaction (PCR)primers are provided or prepared which are sufficiently complementary tothe known loop sequence so as to be hybridizable under PCR conditions,wherein at least one of the primers contains a modified nucleic acidsequence to be inserted into the DNA sequence. PCR is performed usingthe replicatable DNA sequence and the primers. The reaction product ofthe PCR is then expressed so as to yield a polypeptide monobody.

The invention further provides a method of preparing a Fn3 polypeptidemonobody. The method comprises providing a replicatable DNA sequenceencoding a plurality of Fn3 β-strand domain sequences that are linked toa plurality of loop region sequences, wherein the nucleotide sequence ofat least one loop region is known. Site-directed mutagenesis of at leastone loop region is performed so as to create an insertion mutation. Theresultant DNA comprising the insertion mutation is then expressed.

Further provided is a variegated nucleic acid library encoding Fn3polypeptide monobodies comprising a plurality of nucleic acid speciesencoding a plurality of Fn3 β-strand domain sequences that are linked toa plurality of loop region sequences, wherein one or more of themonobody loop region sequences vary by deletion, insertion orreplacement of at least two amino acids from corresponding loop regionsequences in wild-type Fn3, and wherein the β-strand domains of themonobody have at least a 50% total amino acid sequence homology to thecorresponding amino acid sequence of β-strand domain sequences of thewild-type Fn3. The invention also provides a peptide display libraryderived from the variegated nucleic acid library of the invention.Preferably, the peptide of the peptide display library is displayed onthe surface of a bacteriophage, e.g., a M13 bacteriophage or a fdbacteriophage, or virus.

The invention also provides a method of identifying the amino acidsequence of a polypeptide molecule capable of binding to a specificbinding partner (SBP) so as to form a polypeptide:SSP complex, whereinthe dissociation constant of the said polypeptide:SBP complex is lessthan 10⁻⁶ moles/liter. The method comprises the steps of:

-   -   a) providing a peptide display library of the invention;    -   b) contacting the peptide display library of (a) with an        immobilized or separable SBP;    -   c) separating the peptide:SBP complexes from the free peptides;    -   d) causing the replication of the separated peptides of (c) so        as to result in a new peptide display library distinguished from        that in (a) by having a lowered diversity and by being enriched        in displayed peptides capable of binding the SBP;    -   e) optionally repeating steps (b), (c), and (d) with the new        library of (d); and    -   f) determining the nucleic acid sequence of the region encoding        the displayed peptide of a species from (d) and hence deducing        the peptide sequence capable of binding to the SBP.

The present invention also provides a method of preparing a variegatednucleic acid library encoding Fn3 polypeptide monobodies having aplurality of nucleic acid species each comprising a plurality of loopregions, wherein the species encode a plurality of Fn3 β-strand domainsequences that are linked to a plurality of loop region sequences,wherein one or more of the loop region sequences vary by deletion,insertion or replacement of at least two amino acids from correspondingloop region sequences in wild-type Fn3, and wherein the β-strand domainsequences of the monobody have at least a 50% total amino acid sequencehomology to the corresponding amino acid sequences of β-strand domainsequences of the wild-type Fn3, comprising the steps of

-   -   a) preparing an Fn3 polypeptide monobody having a predetermined        sequence;    -   b) contacting the polypeptide with a specific binding partner        (SBP) so as to form a polypeptide:SSP complex wherein the        dissociation constant of the said polypeptide:SBP complex is        less than 10⁻⁶ moles/liter;    -   c) determining the binding structure of the polypeptide:SBP        complex by nuclear magnetic resonance spectroscopy or X-ray        crystallography, and    -   d) preparing the variegated nucleic acid library, wherein the        variegation is performed at positions in the nucleic acid        sequence which, from the information provided in (c), result in        one or more polypeptides with improved binding to the SBP.

Also provided is a method of identifying the amino acid sequence of apolypeptide molecule capable of catalyzing a chemical reaction with acatalyzed rate constant, K_(cat), and an uncatalyzed rate constant,k_(uncat), such that the ratio of k_(cat)/k_(uncat), is greater than 10.The method comprises the steps of:

-   -   a) providing a peptide display library of the invention;    -   b) contacting the peptide display library of (a) with an        immobilized or separable transition state analog compound (TSAC)        representing the approximate molecular transition state of the        chemical reaction;    -   c) separating the peptide:TSAC complexes from the free peptides;    -   d) causing the replication of the separated peptides of (c) so        as to result in a new peptide display library distinguished from        that in (a) by having a lowered diversity and by being enriched        in displayed peptides capable of binding the TSAC;    -   e) optionally repeating steps (b), (c), and (d) with the new        library of (d); and    -   f) determining the nucleic acid sequence of the region encoding        the displayed peptide of a species from (d) and hence deducing        the peptide sequence.

The invention also provides a method of preparing a variegated nucleicacid library encoding Fn3 polypeptide monobodies having a plurality ofnucleic acid species each comprising a plurality of loop regions,wherein the species encode a plurality of Fn3 β-strand domain sequencesthat are linked to a plurality of loop region sequences, wherein one ormore of the loop region sequences vary by deletion, insertion orreplacement of at least two amino acids from corresponding loop regionsequences in wild-type Fn3, and wherein the β-strand domain sequences ofthe monobody have at least a 50% total amino acid sequence homology tothe corresponding amino acid sequences of β-strand domain sequences ofthe wild-type Fn3, comprising the steps of

-   -   a) preparing an Fn3 polypeptide monobody having a predetermined        sequence, wherein the polypeptide is capable of catalyzing a        chemical reaction with a catalyzed rate constant, k_(cat), and        an uncatalyzed rate constant, k_(uncat), such that the ratio of        k_(cat)/k_(uncat) is greater than 10;    -   b) contacting the polypeptide with an immobilized or separable        transition state analog compound (TSAC) representing the        approximate molecular transition state of the chemical reaction;    -   c) determining the binding structure of the polypeptide:TSAC        complex by nuclear magnetic resonance spectroscopy or X-ray        crystallography, and    -   d) preparing the variegated nucleic acid library, wherein the        variegation is performed at positions in the nucleic acid        sequence which, from the information provided in (c), result in        one or more polypeptides with improved binding to or        stabilization of the TSAC.

The invention also provides a kit for the performance of any of themethods of the invention. The invention further provides a composition,e.g., a polypeptide, prepared by the use of the kit, or identified byany of the methods of the invention.

The following abbreviations have been used in describing amino acids,peptides, or proteins: Ala, or A, Alanine; Arg, or R, Arginine; Asn orN, asparagine; Asp, or D, aspartic acid; Cysor C, cystein; Gln, or Q,glutamine; Glu, or E, glutamic acid; Gly, or G, glycine; His, or H,histidine; IIe, or I, isoleucine; Leu, or L, leucine; Lys, or K, lysine;Met, or M, methionine; Phe, or F, phenylalanine; Pro, or P, proline;Ser, or S, serine; Thr, or T, threonine; Trp, or W, tryptophan; Tyr, orY, tyrosine; Val, or V, valine.

The following abbreviations have been used in describing nucleic acids,DNA, or RNA: A, adenosine; T, thymidine; G, guanosine; C, cytosine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. β-Strand and loop topology of anti-lysozyme immunoglobulinD1.3. (Bhat et al., 1994). The locations of complementarity determiningregions (CDRs, hypervariable regions) are indicated.

FIG. 1B. β-Strand and loop topology of the 10th type III domain of humanfibronectin. (Main et al., 1992) The locations of the integrin-bindingArg-Gly-Asp (RGD) sequence is indicated.

FIG. 1C. MOLSCRIPT representation of anti-lysozyme immunoglobulin D1.3.(Fraulis, 1991; Bhat et al., 1994) The locations of complementaritydetermining regions (CDRs, hypervariable regions) are indicated.

FIG. 1D. MOLSCRIPT representation of the 10th type III domain of humanfibronectin. (Kraulis, 1991; Main et al., 1992) The locations of theintegrin-binding Arg-Gly-Asp (RGD) sequence is indicated.

FIG. 2. Amino acid sequence (SEQ ID NO:110) and restriction sites of thesynthetic Fn3 gene. The residue numbering is according to Main et al.(1992). Restriction enzyme sites designed are shown above the amino acidsequence. β-Strands are denoted by underlines. The N-terminal “mq”sequence has been added for a subsequent cloning into an expressionvector. The His-tag (Novagen) fusion protein has an additional sequence,MGSSHHHHHHSSGLVPRGSH (SEQ ID NO: 114), preceding the Fn3 sequence shownabove.

FIG. 3A. Far UV CD spectra of wild-type Fn3 at 25° C. and 90° C. Fn3 (50μM) was dissolved in sodium acetate (50 mM, pH 4.6).

FIG. 3B. Thermal denaturation of Fn3 monitored at 215 μm. Temperaturewas increased at a rate of 1° C./min.

FIG. 4A. Cα trace of the crystal structure of the complex of lysozyme(HEL) and the Fv fragment of the anti-hen egg-white lysozyme (anti-HEL)antibody D1.3 (Bhat et al., 1994). Side chains of the residues 99-102 ofVH CDR3, which make contact with HEL, are also shown.

FIG. 4B. Contact surface area for each residue of the D 1.3 VH-HEL andVH-VL interactions plotted vs. residue number of D1.3 VH. Surface areaand secondary structure were determined using the program DSSP (Kabshand Sander, 1983).

FIG. 4C. Schematic drawings of the β-sheet structure of the Fstrand-loop-G strand moieties of D1.3 VH. The boxes denote residues inβ-strands and ovals those not in strands. The shaded boxes indicateresidues of which side chains are significantly buried. The broken linesindicate hydrogen bonds.

FIG. 4D. Schematic drawings of the β-sheet structure of the Fstrand-loop-G strand moieties of Fn3. The boxes denote residues inβ-strands and ovals those not in strands. The shaded boxes indicateresidues of which side chains are significantly buried. The broken linesindicate hydrogen bonds.

FIG. 5. Designed Fn3 gene showing DNA and amino acid sequences (SEQ IDNO:1111 and SEQ ID NO:112). The amino acid numbering is according toMain et al. (1992). The two loops that were randomized in combinatoriallibraries are enclosed in boxes.

FIG. 6. Map of plasmid pAS45. Plasmid pAS45 is the expression vector ofHis-tag-Fn3.

FIG. 7. Map of plasmid pAS25. Plasmid pAS25 is the expression vector ofFn3.

FIG. 8. Map of plasmid pAS38. pAS38 is a phagmid vector for the surfacedisplay of Fn3.

FIG. 9. (Ubiquitin-1) Characterization of ligand-specific binding ofenriched clones using phage enzyme-linked immunosolvent assay (ELISA).Microtiter plate wells were coated with ubiquitin (1 μg/well; “Ligand(+)) and then blocked with BSA. Phage solution in TBS containingapproximately 10¹⁰ colony forming units (cfu) was added to a well andwashed with TBS. Bound phages were detected with anti-phage antibody-PODconjugate (Pharmacia) with Turbo-TMB (Pierce) as a substrate. Absorbancewas measured using a Molecular Devices SPECTRAMAX 250 microplatespectrophotometer. For a control, wells without the immobilized ligandwere used. 2-1 and 2-2 denote enriched clones from Library 2 eluted withfree ligand and acid, respectively. 4-1 and 4-2 denote enriched clonesfrom Library 4 eluted with free ligand and acid, respectively.

FIG. 10. (Ubiquitin-2) Competition phage ELISA of enriched clones. Phagesolutions containing approximately 10¹⁰ cfu were first incubated withfree ubiquitin at 4° C. for 1 hour prior to the binding to aligand-coated well. The wells were washed and phages detected asdescribed above.

FIG. 11. Competition phage ELISA of ubiquitin-binding monobody 411.Experimental conditions are the same as described above for ubiquitin.The ELISA was performed in the presence of free ubiquitin in the bindingsolution. The experiments were performed with four differentpreparations of the same clone.

FIG. 12. (Fluorescein-1) Phage ELISA of four clones, pLB25.1 (containingSEQ ID NO:115), pLB25.4 (containing SEQ ID NO:116), pLB24.1 (containingSEQ ID NO:117) and pLB24.3 (containing SEQ ID NO:118). Experimentalconditions are the same as ubiquitin-1 above.

FIG. 13. (Fluorescein-2) Competition ELISA of the four clones.Experimental conditions are the same as ubiquitin-2 above.

FIG. 14. ¹H, ¹⁵N—HSQC spectrum of a fluorescence-binding monobodyLB25.5. Approximately 20 μm protein was dissolved in 10 mm sodiumacetate buffer (pH 5.0) containing 100 mM sodium chloride. The spectrumwas collected at 30° C. on a Varian Unity INOVA 600 NMR spectrometer.

FIG. 15A. Characterization of the binding reaction of Ubi4-Fn3 to thetarget, ubiquitin. Phage ELISA analysis of binding of Ubi4-Fn3 toubiquitin. The binding of Ubi4-phages to ubiquitin-coated wells wasmeasured. The control experiment was performed with wells containing noubiquitin.

FIG. 15B. Competition phage ELISA of Ubi4-Fn3. Ubi4-Fn3 phages werepreincubated with soluble ubiquitin at an indicated concentration,followed by the phage ELISA detection in ubiquitin-coated wells.

FIG. 15C. Competition phage ELISA testing the specificity of the Ubi4clone. The Ubi4 phages were preincubated with 250 μg/ml of solubleproteins, followed by phage ELISA as in (b).

FIG. 15D. ELISA using free proteins.

FIG. 16. Equilibrium unfolding curves for Ubi4-Fn3 (closed symbols) andwild-type Fn3 (open symbols). Squares indicate data measured in TBS(Tris HCl buffer (50 mM, pH 7.5) containing NaCl (150 mM)). Circlesindicate data measured in Gly HCl buffer (20 mM, pH 3.3) containing NaCl(300 mM). The curves show the best fit of the transition curve based onthe two-state model. Parameters characterizing the transitions arelisted in Table 7.

FIG. 17. (a) ¹H, ¹⁵N-HSQC spectrum of [¹⁵N]-Ubi4-K Fn3. (b). Difference(δ_(wild-type)-δ_(Ubi4)) of ¹H (b) and ¹⁵N (c) chemical shifts plottedversus residue number. Values for residues 82-84 (shown as filledcircles) where Ubi4-K deletions are set to zero. Open circles indicateresidues that are mutated in the Ubi4-K protein. The locations ofβ-strands are indicated with arrows.

DETAILED DESCRIPTION OF THE INVENTION

For the past decade the immune system has been exploited as a richsource of de novo catalysts. Catalytic antibodies have been shown tohave chemoselectivity, enantioselectivity, large rate accelerations, andeven an ability to reroute chemical reactions. In most cases theantibodies have been elicited to transition state analog (TSA) haptens.These TSA haptens are stable, low-molecular weight compounds designed tomimic the structures of the energetically unstable transition statespecies that briefly (approximate half-life 10⁻¹³ s) appear alongreaction pathways between reactants and products. Anti-TSA antibodies,like natural enzymes, are thought to selectively bind and stabilizetransition state, thereby easing the passage of reactants to products.Thus, upon binding, the antibody lowers the energy of the actualtransition state and increases the rate of the reaction. These catalystscan be programmed to bind to geometrical and electrostatic features ofthe transition state so that the reaction route can be controlled byneutralizing unfavorable charges, overcoming entropic barriers, anddictating stereoelectronic features of the reaction. By this means evenreactions that are otherwise highly disfavored have been catalyzed(Janda et al. 1997). Further, in many instances catalysts have been madefor reactions for which there are no known natural or manmade enzymes.

The success of any combinatorial chemical system in obtaining aparticular function depends on the size of the library and the abilityto access its members. Most often the antibodies that are made in ananimal against a hapten that mimics the transition state of a reactionare first screened for binding to the hapten and then screened again forcatalytic activity. An improved method allows for the direct selectionfor catalysis from antibody libraries in phage, thereby linkingchemistry and replication.

A library of antibody fragments can be created on the surface offilamentous phage viruses by adding randomized antibody genes to thegene that encodes the phage's coat protein. Each phage then expressesand displays multiple copies of a single antibody fragment on itssurface. Because each phage possesses both the surface-displayedantibody fragment and the DNA that encodes that fragment, and antibodyfragment that binds to a target can be identified by amplifying theassociated DNA.

Immunochemists use as antigens materials that have as little chemicalreactivity as possible. It is almost always the case that one wishes theultimate antibody to interact with native structures. In reactiveimmunization the concept is just the opposite. One immunizes withcompounds that are highly reactive so that upon binding to the antibodymolecule during the induction process, a chemical reaction ensues. Laterthis same chemical reaction becomes part of the mechanism of thecatalytic event. In a certain sense one is immunizing with a chemicalreaction rather than a substance per se. Reactive immunogens can beconsidered as analogous to the mechanism-based inhibitors thatenzymologists use except that they are used in the inverse way in that,instead of inhibiting a mechanism, they induce a mechanism.

Man-made catalytic antibodies have considerable commercial potential inmany different applications. Catalytic antibody-based products have beenused successfully in prototype experiments in therapeutic applications,such as prodrug activation and cocaine inactivation, and innontherapeutic applications, such as biosensors and organic synthesis.

Catalytic antibodies are theoretically more attractive than noncatalyticantibodies as therapeutic agents because, being catalytic, they may beused in lower doses, and also because their effects are unusuallyirreversible (for example, peptide bond cleavage rather than binding).In therapy, purified catalytic antibodies could be directly administeredto a patient, or alternatively the patient's own catalytic antibodyresponse could be elicited by immunization with an appropriate hapten.Catalytic antibodies also could be used as clinical diagnostic tools oras regioselective or stereoselective catalysts in the synthesis of finechemicals.

I. Mutation of Fn3 Loops and Grafting of Ab Loops onto Fn3

An ideal scaffold for CDR grafting is highly soluble and stable. It issmall enough for structural analysis, yet large enough to accommodatemultiple CDRs so as to achieve tight binding and/or high specificity.

A novel strategy to generate an artificial Ab system on the framework ofan existing non-Ab protein was developed. An advantage of this approachover the minimization of an Ab scaffold is that one can avoid inheritingthe undesired properties of Abs. Fibronectin type m domain (Fn3) wasused as the scaffold. Fibronectin is a large protein which playsessential roles in the formation of extracellular matrix and cell-cellinteractions; it consists of many repeats of three types (II and II) ofsmall domains (Baron et al., 1991). Fn3 itself is the paradigm of alarge subfamily (Fn3 family or s-type Ig family) of the immunoglobulinsuperfamily (IgSF). The Fn3 family includes cell adhesion molecules,cell surface hormone and cytokine receptors, chaperoning, andcarbohydrate-binding domains (for reviews, see Bork & Doolittle, 1992;Jones, 1993; Bork et al., 1994; Campbell & Spitzfaden, 1994; Harpez &Chothia, 1994).

Recently, crystallographic studies revealed that the structure of theDNA binding domains of the transcription factor NF-kB is also closelyrelated to the Fn3 fold (Ghosh et al., 1995; Müller et al., 1995). Theseproteins are all involved in specific molecular recognition, and in mostcases ligand-binding sites are formed by surface loops, suggesting thatthe Fn3 scaffold is an excellent framework for building specific bindingproteins. The 3D structure of Fn3 has been determined by NMR (Main etal., 1992) and by X-ray crystallography (Leahy et al., 1992; Dickinsonet al., 1994). The structure is best described as a β-sandwich similarto that of Ab VH domain except that Fn3 has seven β-strands instead ofnine (FIG. 1). There are three loops on each end of Fn3; the positionsof the BC, DE and FG loops approximately correspond to those of CDR1, 2and 3 of the VH domain, respectively (FIG. 1 C, D).

Fn3 is small (˜95 residues), monomeric, soluble and stable. It is one offew members of IgSF that do not have disulfide bonds; VH has aninterstrand disulfide bond (FIG. 1A) and has marginal stability underreducing conditions. Fn3 has been expressed in E. coli (Aukhil et al.,1993). In addition, 17 Fn3 domains are present just in humanfibronectin, providing important information on conserved residues whichare often important for the stability and folding (for sequencealignment, see Main et al., 1992 and Dickinson et al., 1994). Fromsequence analysis, large variations are seen in the BC and FG loops,suggesting that the loops are not crucial to stability. NMR studies haverevealed that the FG loop is highly flexible; the flexibility has beenimplicated for the specific binding of the 10th Fn3 to α₅β₁ integrinthrough the Arg-Gly-Asp (RGD) (SEQ ID NO: 113) motif In the crystalstructure of human growth hormone-receptor complex (de Vos et al.,1992), the second Fn3 domain of the receptor interacts with hormone viathe FG and BC loops, suggesting it is feasible to build a binding siteusing the two loops.

The tenth type III module of fibronectin has a fold similar to that ofimmunoglobulin domains, with seven β strands forming two antiparallel βsheets, which pack against each other (Main et al., 1992). The structureof the type II module consists of seven β strands, which form a sandwichof two antiparallel β sheets, one containing three strands (ABE) and theother four strands (C'CFG) (Williams et al., 1988). The triple-strandedβ sheet consists of residues Glu-9-Thr-14 (A), Ser-17-Asp-23 (B), andThr-56-Ser-60 (E). The majority of the conserved residues contribute tothe hydrophobic core, with the invariant hydrophobic residues Trp22 andTry-68 lying toward the N-terminal and C-terminal ends of the core,respectively. The β strands are much less flexible and appear to providea rigid framework upon which functional, flexible loops are built. Thetopology is similar to that of immunoglobulin C domains.

Gene Construction and Mutagenesis

A synthetic gene for tenth Fn3 of human fibronectin (FIG. 2) wasdesigned which includes convenient restriction sites for ease ofmutagenesis and uses specific codons for high-level protein expression(Gribskov et al., 1984).

The gene was assembled as follows: (1) the gene sequence was dividedinto five parts with boundaries at designed restriction sites (FIG. 2);(2) for each part, a pair of oligonucleotides that code opposite strandsand have complementary overlaps of ˜15 bases was synthesized; (3) thetwo oligonucleotides were annealed and single strand regions were filledin using the Klenow fragment of DNA polymerase; (4) the double-strandedoligonucleotide was cloned into the pET3a vector (Novagen) usingrestriction enzyme sites at the termini of the fragment and its sequencewas confirmed by an Applied Biosystems DNA sequencer using the dideoxytermination protocol provided by the manufacturer, (5) steps 2-4 wererepeated to obtain the whole gene (plasmid pAS25) (FIG. 7).

Although the present method takes more time to assemble a gene than theone-step polymerase chain reaction (PCR) method (Sandhu et al., 1992),no mutations occurred in the gene. Mutations would likely have beenintroduced by the low fidelity replication by Taq polymerase and wouldhave required time-consuming gene editing. The gene was also cloned intothe pET15b (Novagen) vector (pEW1). Both vectors expressed the Fn3 geneunder the control of bacteriophage T7 promoter (Studler et al. 1990);pAS25 expressed the 96-residue Fn3 protein only, while pEW1 expressedFn3 as a fusion protein with poly-histidine peptide (His•tag).Recombinant DNA manipulations were performed according to MolecularCloning (Sambrook et al., 1989), unless otherwise stated.

Mutations were introduced to the Fn3 gene using either cassettemutagenesis or oligonucleotide site-directed mutagenesis techniques(Deng & Nickoloff, 1992). Cassette mutagenesis was performed using thesame protocol for gene construction described above; double-stranded DNAfragment coding a new sequence was cloned into an expression vector(pAS25 and/or pEW1). Many mutations can be made by combining a newlysynthesized strand (coding mutations) and an oligonucleotide used forthe gene synthesis. The resulting genes were sequenced to confirm thatthe designed mutations and no other mutations were introduced bymutagenesis reactions.

Design and Synthesis of Fn3 Mutants with Antibody CDRs

Two candidate loops (FG and BC) were identified for grafting Antibodieswith known crystal structures were examined in order to identifycandidates for the sources of loops to be grafted onto Fn3. Anti-hen egglysozyme (HEL) antibody D1.3 (Bhat et al., 1994) was chosen as thesource of a CDR loop. The reasons for this choice were: (1) highresolution crystal structures of the free and complexed states areavailable (FIG. 4 A; Bhat et al., 1994), (2) thermodynamics data for thebinding reaction are available (Tello et al., 1993), (3) D1.3 has beenused as a paradigm for Ab structural analysis and Ab engineering(Verhoeyen et al., 1988; McCafferty et al., 1990) (4) site-directedmutagenesis experiments have shown that CDR3 of the heavy chain(VH-CDR3) makes a larger contribution to the affinity than the otherCDRs (Hawkins et al., 1993), and (5) a binding assay can be easilyperformed. The objective for this trial was to graft VH-CDR3 of D 1.3onto the Fn3 scaffold without significant loss of stability.

An analysis of the D1.3 structure (FIG. 4) revealed that only residues99-102 (“RDYR”) make direct contact with hen egg-white lysozyme (HEL)(FIG. 4 B), although VH-CDR3 is defined as longer (Bhat et al., 1994).It should be noted that the C-terminal half of VH-CDR3 (residues101-104) made significant contact with the VL domain (FIG. 4 B). It hasalso become clear that D1.3 VH-CDR3 (FIG. 4 C) has a shorter turnbetween the strands F and G than the FG loop of Fn3 (FIG. 4 D).Therefore, mutant sequences were designed by using the RDYR (99-102) ofD1.3 as the core and made different boundaries and loop lengths (Table1). Shorter loops may mimic the D 1.3 CDR3 conformation better, therebyyielding higher affinity, but they may also significantly reducestability by removing wild-type interactions of Fn3. TABLE 1 Amino acidsequences of D 1.3 VII CDR3, VH8 CDR3 and Fn3 FG loop and list ofplanned mutants. 96     100         105 •       •           • D1.3A R E R D Y R L D Y W G Q G (SEQ ID NO:1) VH8 A R G A V V S Y Y AM D Y W G Q G (SEQ ID NO:2)      75        80       85      •        •        • Fn3 Y A V T G R G D S P A S S K P I (SEQ IDNO:3) Mutant Sequence D1.3-1 Y A E R D Y R L D Y - - - - P I (SEQ IDNO:4) D1.3-2 Y A V R D Y R L D Y - - - - P I (SEQ ID NO:5) D1.3-3 Y A VR D Y R L D Y A S S K P I (SEQ ID NO:6) D1.3-4 Y A V R D Y R L D Y - - -K P I (SEQ ID NO:7) D1.3-5 Y A V R D Y R - - - - - S K P I (SEQ ID NO:8)D1.3-6 Y A V T R D Y R L - - S S K P I (SEQ ID NO:9) D1.3-7 Y A V T E RD Y R L - S S K P I (SEQ ID NO:10) VH8-1 Y A V A V V S Y Y A M D Y - P I(SEQ ID NO:11) VH8-2 Y A V T A V V S Y Y A S S K P I (SEQ ID NO:12)Underlines indicate residues in β-strands. Bold characters indicatereplaced residues.

In addition, an anti-HEL single VH domain termed VH8 (Ward et al., 1989)was chosen as a template. VH8 was selected by library screening and, inspite of the lack of the VL domain, VH8 has an affinity for HEL of 27nM, probably due to its longer VH-CDR3 (Table 1). Therefore, its VH-CDR3was grafted onto Fn3. Longer loops may be advantageous on the Fn3framework because they may provide higher affinity and also are close tothe loop length of wild-type Fn3. The 3D structure of VH8 was not knownand thus the VH8 CDR3 sequence was aligned with that of D1.3 VH-CDR3;two loops were designed (Table 1).

Mutant Construction and Production

Site-directed mutagenesis experiments were performed to obtain designedsequences. Two mutant Fn3s, D1.3-1 and D1.3-4 (Table 1) were obtainedand both were expressed as soluble His-tag fusion proteins. D1.3-4 waspurified and the His.tag portion was removed by thrombin cleavage.D1.3-4 is soluble up to at least 1 mM at pH 7.2. No aggregation of theprotein has been observed during sample preparation and NMR dataacquisition.

Protein Expression and Purification

E. coli BL21 (DE3) (Novagen) were transformed with an expression vector(pAS25, pEW1 and their derivatives) containing a gene for the wild-typeor a mutant Cells were grown in M9 minimal medium and M9 mediumsupplemented with Bactotrypton (Difco) containing ampicillin (200μg/ml). For isotopic labeling, ¹⁵N NH⁴CI and/or ¹³C glucose replacedunlabeled components. 500 mL medium in a 2 liter baffle flask wereinoculated with 10 ml of overnight culture and agitated at 37° C.Isopropylthio-β-galactoside (IPTG) was added at a final concentration of1 mM to initiate protein expression when OD (600 nm) reaches one. Theceils were harvested by centrifugation 3 hours after the addition ofIPTG and kept frozen at −70° C. until used.

Fn3 without His•tag was purified as follows. Cells were suspended in 5ml/(g cell) of Tris (50 mM, pH 7.6) containingethylenediaminetetraacetic acid (EDTA; 1 mM) and phenylmethylsulfonylfluoride (1 mM). HEL was added to a final concentration of 0.5 mg/mLAfter incubating the solution for 30 minutes at 37° C., it was sonicatedthree times for 30 seconds on ice. Cell debris was removed bycentrifugation. Ammonium sulfate was added to the solution andprecipitate recovered by centrifugation. The pellet was dissolved in5-10 ml sodium acetate (50 mM, pH 4.6) and insoluble material wasremoved by centrifugation. The solution was applied to a SEPHACRYLS100HR column (Pharmacia) equilibrated in the sodium acetate buffer.Fractions containing Fn3 then was applied to a RESOURCES® column(Pharmacia) equilibrated in sodium acetate (50 mM, pH 4.6) and elutedwith a linear gradient of sodium chloride (0-0.5 M). The protocol can beadjusted to purify mutant proteins with different surface chargeproperties.

Fn3 with His•tag was purified as follows. The soluble fraction wasprepared as described above, except that sodium phosphate buffer (50 mM,pH 7.6) containing sodium chloride (100 mM) replaced the Tris buffer.The solution was applied to a HI-TRAP chelating column (Pharmacia)preloaded with nickel and equilibrated in the phosphate buffer. Afterwashing the column with the buffer, His•tag-Fn3 was eluted in thephosphate buffer containing 50 mM EDTA Fractions containing His•tag-Fn3were pooled and applied to a SEPHACRYL S100-HR column, yielding highlypure protein. The His•tag portion was cleaved off by treating the fusionprotein with thrombin using the protocol supplied by Novagen. Fn3 wasseparated from the His•tag peptide and thrombin by a RESOURCES® columnusing the protocol above.

The wild-type and two mutant proteins so far examined are expressed assoluble proteins. In the case that a mutant is expressed as inclusionbodies (insoluble aggregate), it is first examined if it can beexpressed as a soluble protein at lower temperature (e.g., 25-30° C.).If this is not possible, the inclusion bodies are collected by low-speedcentrifugation following cell lysis as described above. The pellet iswashed with buffer, sonicated and centrifuged. The inclusion bodies aresolubilized in phosphate buffer (50 mM, pH 7.6) containing guanidiniumchloride (GdnCl, 6 M) and will be loaded on a HI-TRAP chelating column.The protein is eluted with the buffer containing GdnCl and 50 mM EDTA.

Conformation of Mutant Fn3, D13-4

The ¹H NMR spectra of His•tag D1.3-4 fusion protein closely resembledthat of the wild-type, suggesting the mutant is folded in a similarconformation to that of the wild-type. The spectrum of D1.3-4 after theremoval of the His-tag peptide showed a large spectral dispersion. Alarge dispersion of amide protons (7-9.5 ppm) and a large number ofdownfield (5.0-6.5 ppm) C^(α) protons are characteristic of a β-sheetprotein (Wüthrich, 1986).

The 2D NOESY spectrum of D1.3-4 provided further evidence for apreserved conformation. The region in the spectrum showed interactionsbetween upfield methyl protons (<0.5 ppm) and methyl-methylene protons.The Val72 γ methyl resonances were well separated in the wild-typespectrum (−0.07 and 0.37 ppm; (Baron et al., 1992)). Resonancescorresponding to the two methyl protons are present in the D1.3-4spectrum (−0.07 and 0.44 ppm). The cross peak between these tworesonances and other conserved cross peaks indicate that the tworesonances in the D1.3-4 spectrum are highly likely those of Val72 andthat other methyl protons are in nearly identical environment to that ofwild-type Fn3. Minor differences between the two spectra are presumablydue to small structural perturbation due to the mutations. Va172 is onthe F strand, where it forms a part of the central hydrophobic core ofFn3 (Main et al., 1992). It is only four residues away from the mutatedresidues of the FG loop (Table 1). The results are remarkable because,despite there being 7 mutations and 3 deletions in the loop (more than10% of total residues; FIG. 12, Table 2), D1.3-4 retains a 3D structurevirtually identical to that of the wild-type (except for the mutatedloop). Therefore, the results provide strong support that the FG loop isnot significantly contributing to the folding and stability of the Fn3molecule and thus that the FG loop can be mutated extensively. TABLE 2Sequences of oligonucleotides Name Sequence FN1FCGGGATCCCATATGCAGGTTTCTGATGTTCCGCGTGACCTGGAAGTTGTTGCTGCGACC (SEQ ID NO:13) FN1R TAACTGCAGGAGCATCCCAGCTGATCAGCAGGCTAGTCGGGGTCGCAGCAACAAC (SEQ IDNO:14) FN2F CTCCTGCAGTTACCGTGCGTTATTACCGTATCACGTACGGTGAAACCGGTG (SEQ IDNO: 15) FN2R GTGAATTCCTGAACCGGGGAGTTACCACCGGTTTCACCG (SEQ ID NO:16) FN3FAGGAATTCACTGTACCTGGTTCCAAGTCTACTGCTACCATCAGCGG (SEQ ID NO:17) FN3RGTATAGTCGACACCCGGTTTCAGGCCGCTGATGGTAGC (SEQ ID NO:18) FN4FCGGGTGTCGACTATACCATCACTGTATACGCT (SEQ ID NO:19) FN4RCGGGATCCGAGCTCGCTGGGCTGTCACCACGGCCAGTAACAGCGTATACAGTGAT (SEQ ID NO: 20)FN5F CAGCGAGCTCCAAGCCAATCTCGATTAACTACCGT (SEQ ID NO:21) FN5RCGGGATCCTCGAGTTACTAGGTACGGTAGTTAATCGA (SEQ ID NO:22) FN5R40CGGGATCCACGCGTGCCACCGGTACGGTAGTTAATCGA (SEQ ID NO:23) qene3FCGGGATCCACGCGTCCATTCGTTTGTGAATATCAAGGCCAATCG (SEQ ID NO:24) gene3RCCGGAAGCTTTAAGACTCCTTATTACGCAGTATGTTAGC (SEQ ID NO:25) 38TAABg1IICTGTTACTGGCCGTGAGATCTAACCAGCGAGCTCCA (SEQ ID NO:26) Bc3GATCAGCTGGGATGCTCCTNNKNNKNNKNNKNNKTATTACCGTATCACGTA (SEQ ID NO: 27) FG2TGTATACGCTGTTACTGGCNNKNNKNNKNNKNNKNNKNNKTTCCAAGCCAATCTCGAT (SEQ ID NO:28) FG3 CTGTATACGCTGTTACTGGCNNKNNKNNKNNKCCAGCGAGCTCCAAG (SEQ ID NO:29)FG4 CATCACTGTATACGCTGTTACTNNKNNKNNKNNKNNKTCCAAGCCAATCTC (SEQ ID NO: 30)Restriction enzyme sites are underlined. N and K denote an equimolarmixture of A, T. G and C and that of G and T, respectively.Structure and Stability Measurements

Structures of Abs were analyzed using quantitative methods (e.g., DSSP(Kabsch & Sander, 1983) and PDBFIT (D. McRee, The Scripps ResearchInstitute)) as well as computer graphics (e.g., QUANTA (MolecularSimulations) and WHAT IF (G. Vriend, European Molecular BiologyLaboratory)) to superimpose the strand-loop-strand structures of Abs andFn3.

The stability of FnAbs was determined by measuring temperature- andchemical denaturant-induced unfolding reactions (Pace et al., 1989). Thetemperature-induced unfolding reaction was measured using a circulardichroism (CD) polarimeter. Ellipticity at 222 and 215 nm was recordedas the sample temperature was slowly raised. Sample concentrationsbetween −10 and 50 μM were used. After the unfolding baseline wasestablished, the temperature was lowered to examine the reversibility ofthe unfolding reaction. Free energy of unfolding was determined byfitting data to the equation for the two-state transition (Becktel &Schellman, 1987; Pace et al., 1989). Nonlinear least-squares fitting wasperformed using the program IGOR (WaveMetrics) on a Macintosh computer.

The structure and stability of two selected mutant Fn3s were studied;the first mutant was D1.3-4 (Table 2) and the second was a mutant calledAS40 which contains four mutations in the BC loop (A²⁶V²⁷T²⁸V²⁹)→TQRQ).AS40 was randomly chosen from the BC loop library described above. Bothmutants were expressed as soluble proteins in E. coli and wereconcentrated at least to 1 mM, permitting NMR studies.

The mid-point of the thermal denaturation for both mutants wasapproximately 69° C., as compared to approximately 79° C. for thewild-type protein. The results indicated that the extensive mutations atthe two surface loops did not drastically decrease the stability of Fn3,and thus demonstrated the feasibility of introducing a large number ofmutations in both loops.

Stability was also determined by guanidinium chloride (GdnCl)- andurea-induced unfolding reactions. Preliminary unfolding curves wererecorded using a fluorometer equipped with a motor-driven syringe; GdnClor urea were added continuously to the protein solution in the cuvette.Based on the preliminary unfolding curves, separate samples containingvarying concentration of a denaturant were prepared and fluorescence(excitation at 290 nm, emission at 300-400 nm) or CD (ellipticity at 222and 215 mm) were measured after the samples were equilibrated at themeasurement temperature for at least one hour. The curve was fitted bythe least-squares method to the equation for the two-state model(Santoro & Bolen, 1988; Koide et al., 1993). The change in proteinconcentration was compensated if required.

Once the reversibility of the thermal unfolding reaction is established,the unfolding reaction is measured by a Microcal MC-2 differentialscanning calorimeter (DSC). The cell (˜1.3 ml) will be filled with FnAbsolution (0.1-1 mM) and ΔCp(=ΔH/ΔT) will be recorded as the temperatureis slowly raised. T_(m) (the midpoint of unfolding), ΔH of unfolding andΔG of unfolding is determined by fitting the transition curve (Privalov& Potekhin, 1986) with the ORIGIN software provided by Microcal.

Thermal Unfolding

A temperature-induced unfolding experiment on Fn3 was performed usingcircular dichroism (CD) spectroscopy to monitor changes in secondarystructure. The CD spectrum of the native Fn3 shows a weak signal near222 nm (FIG. 3A), consistent with the predominantly β-structure of Fn3(Perczel et al., 1992). A cooperative unfolding transition is observedat 80-90° C., clearly indicating high stability of Fn3 (FIG. 3B). Thefree energy of unfolding could not be determined due to the lack of apost-transition baseline. The result is consistent with the highstability of the first Fn3 domain of human fibronectin (Litvinovich etal., 1992), thus indicating that Fn3 domains are in general highlystable.

Binding Assays

Binding reaction of FnAbs were characterized quantitatively using anisothermal titration calorimeter (ITC) and fluorescence spectroscopy.

The enthalpy change (ΔH) of binding were measured using a Microcal OMEGAITC (Wiseman et al., 1989). The sample cell (˜1.3 ml) was filled withFnAbs solution (≦100 μM, changed according to K_(d)), and the referencecell filled with distilled water, the system was equilibrated at a giventemperature until a stable baseline is obtained; 5-20 μl of ligandsolution (≦2 mM) was injected by a motordriven syringe within a shortduration (20 sec) followed by an equilibration delay (4 minutes); theinjection was repeated and heat generation/absorption for each injectionwas measured. From the change in the observed heat change as a functionof ligand concentration, ΔH and K_(d) was determined (Wiseman et al.,1989). ΔG and ΔS of the binding reaction was deduced from the twodirectly measured parameters. Deviation from the theoretical curve wasexamined to assess nonspecific (multiplesite) binding. Experiments werealso performed by placing a ligand in the cell and titrating with anFnAb. It should be emphasized that only ITC gives direct measurement ofΔH, thereby malting it possible to evaluate enthalpic and entropiccontributions to the binding energy. ITC was successfully used tomonitor the binding reaction of the D1.3 Ab (Tello et al., 1993; Bhat etal., 1994).

Intrinsic fluorescence is monitored to measure binding reactions withK_(d) in the sub-μM range where the determination of K_(d) by ITC isdifficult Trp fluorescence (excitation at ˜290 nm, emission at 300-350nm) and Tyr fluorescence (excitation at 260 nm, emission at ˜303 nm) ismonitored as the Fn3-mutant solution (≦10 μM) is titrated with ligandsolution (≦100 M). K_(d) of the reaction is determined by the nonlinearleast-squares fitting of the bimolecular binding equation. Presence ofsecondary binding sites is examined using Scatchard analysis. In allbinding assays, control experiments are performed busing wild-type Fn3(or unrelated FnAbs) in place of FnAbs of interest.

II. Production of Fn3 Mutants with High Affinity and Specificity FnAbs

Library screening was carried out in order to select FnAbs which bind tospecific ligands. This is complementary to the modeling approachdescribed above. The advantage of combinatorial screening is that onecan easily produce and screen a large number of variants (≧10⁸), whichis not feasible with specific mutagenesis (“rational design”)approaches. The phage display technique (Smith, 1985; O'Neil & Hoess,1995) was used to effect the screening processes. Fn3 was fused to aphage coat protein (pHI) and displayed on the surface of filamentousphages. These phages harbor a single-stranded DNA genome that containsthe gene coding the Fn3 fusion protein. The amino acid sequence ofdefined regions of Fn3 were randomized using a degenerate nucleotidesequence, thereby constructing a library. Phages displaying Fn3 mutantswith desired binding capabilities were selected in vitro, recovered andamplified. The amino acid sequence of a selected clone can be identifiedreadily by sequencing the Fn3 gene of the selected phage. The protocolsof Smith (Smith & Scott, 1993) were followed with minor modifications.

The objective was to produce FnAbs which have high affinity to smallprotein ligands. HEL and the B1 domain of staphylococcal protein G(hereafter referred to as protein G) were used as ligands. Protein G issmall (56 amino acids) and highly stable (Minor & Kim, 1994; Smith etal., 1994). Its structure was determined by NMR spectroscopy (Gronenbomet al., 1991) to be a helix packed against a four-strand β-sheet Theresulting FnAb-protein G complexes (−150 residues) is one of thesmallest protein-protein complexes produced to date, well within therange of direct NMR methods. The small size, the high stability andsolubility of both components and the ability to label each with stableisotopes (¹³C and ¹⁵N; see below for protein G) make the complexes anideal model system for NMR studies on protein-protein interactions.

The successful loop replacement of Fn3 (the mutant D1.34) demonstratethat at least ten residues can be mutated without the loss of the globalfold Based on this, a library was first constructed in which onlyresidues in the FG loop are randomized After results of loop replacementexperiments on the BC loop were obtained, mutation sites were extendedthat include the BC loop and other sites.

Construction of Fn3 Phage Display System

An M13 phage-based expression vector pASM1 has been constructed asfollows: an oligonucleotide coding the signal peptide of OmpT was clonedat the 5′ end of the Fn3 gene; a gene fragment coding the C-terminaldomain of M13 pIII was prepared from the wild-type gene III gene of M13mp18 using PCR (Corey et al., 1993) and the fragment was inserted at the3′ end of the OmpT-Fn3 gene; a spacer sequence has been inserted betweenFn3 and pIII. The resultant fragment (OmpTFn3-pIII) was cloned in themultiple cloning site of M13 mp18, where the fusion gene is under thecontrol of the lac promoter. This system will produce the Fn3-pIIIfusion protein as well as the wild-type pIII protein. The co-expressionof wild-type pIII is expected to reduce the number of fusion pIIIprotein, thereby increasing the phage infectivity (Corey et al., 1993)(five copies of pIII are present on a phage particle). In addition, asmaller number of fusion pIII protein may be advantageous in selectingtight binding proteins, because the chelating effect due to multiplebinding sites should be smaller than that with all five copies of fusionpIII (Bass et al., 1990). This system has successfully displayed theserine protease trypsin (Corey et al., 1993). Phages were produced andpurified using E. coli K91kan (Smith & Scott, 1993) according to astandard method (Sambrook et al., 1989) except that phage particles werepurified by a second polyethylene glycol precipitation and acidprecipitation.

Successful display of Fn3 on fusion phages has been confirmed by ELISAusing an Ab against fibronectin (Sigma), clearly indicating that it isfeasible to construct libraries using this system.

An alternative system using the fUSE5 (Parmley & Smith, 1988) may alsobe used The Fn3 gene is inserted to fUSE5 using the SfiI restrictionsites introduced at the 5′- and 3′-ends of the Fn3 gene PCR This systemdisplays only the fusion pIII protein (up to five copies) on the surfaceof a phage. Phages are produced and purified as described-(Smith &Scott, 1993). This system has been used to display many proteins and isrobust The advantage of fUSE5 is its low toxicity. This is due to thelow copy number of the replication form (RF) in the host, which in turnmakes it difficult to prepare a sufficient amount of RF for libraryconstruction (Smith & Scott, 1993).

Construction of Libraries

The first library was constructed of the Fn3 domain displayed on thesurface of MB phage in which seven residues (77-83) in the FG loop (FIG.4D) were randomized Randomization will be achieved by the use of anoligonucleotide containing degenerated nucleotide sequence. Adouble-stranded nucleotide was prepared by the same protocol as for genesynthesis (see above) except that one strand had an (NNK)₆(NNG) sequenceat the mutation sites, where N corresponds to an equimolar mixture of A,T, G and C and K corresponds to an equimolar mixture of G and T. The(NNG) codon at residue 83 was required to conserve the SacI restrictionsite (FIG. 2). The (NNK) codon codes all of the 20 amino acids, whilethe NNG codon codes 14. Therefore, this library contained ˜10⁹independent sequences. The library was constructed by ligating thedouble-stranded nucleotide into the wild-type phage vector, pASM1, andthe transfecting E. coli XL1 blue (Stratagene) using electroporation.XL1 blue has the lacI^(q) phenotype and thus suppresses the expressionof the Fn3-pIII fusion protein in the absence of lac inducers. Theinitial library was propagated in this way, to avoid selection againsttoxic Fn3-pIII clones. Phages displaying the randomized Fn3-pIII fusionprotein were prepared by propagating phages with K91kan as the hostK91kan does not suppress the production of the fusion protein, becauseit does not have lacI^(q) Another library was also generated in whichthe BC loop (residues 26-20) was randomized.

Selection of Displayed FnAbs

Screening of Fn3 phage libraries was performed using the biopanningprotocol (Smith & Scott, 1993); a ligand is biotinylated and the strongbiotinstreptavidin interaction was used to immobilize the ligand on astreptavidin-coated dish Experiments were performed at room temperature(˜22° C.). For the initial recovery of phages from a library, 10 μg of abiotinylated ligand were immobilized on a streptavidin-coatedpolystyrene dish (35 mm, Falcon 1008) and then a phage solution(containing ˜10¹¹ pfu (plaque-forming unit)) was added. After washingthe dish with an appropriate buffer (typically TBST, Tris-HCl (50 mM, pH7.5), NaCl (150 mM) and Tween 20 (0.5%)), bound phages were eluted byone or combinations of the following conditions: low pH, an addition ofa free ligand, urea (up to 6 M) and, in the case of anti-protein GFnAbs, cleaving the protein G-biotin linker by thrombin. Recoveredphages were amplified using the standard protocol using K91kan as thehost (Sambrook et al., 1989). The selection process were repeated 3-5times to concentrate positive clones. From the second round on, theamount of the ligand were gradually decreased (to ˜1 μg) and thebiotinylated ligand were mixed with a phage solution before transferringa dish (G. P. Smith, personal communication). After the final round,10-20 clones were picked, and their DNA sequence will be determined. Theligand affinity of the clones were measured first by the phage-ELISAmethod (see below).

To suppress potential binding of the Fn3 framework (background binding)to a ligand, wild-type Fn3 may be added as a competitor in the buffers.In addition, unrelated proteins (e.g., bovine serum albumin, cytochromec and RNase A) may be used as competitors to select highly specificFnAbs.

Binding Assay

The binding affinity of FnAbs on phage surface is characterizedsemiquantitatively using the phage ELISA technique (Li et al., 1995).Wells of microtiter plates (Nunc) are coated with a ligand protein (orwith streptavidin followed by the binding of a biotinylated ligand) andblocked with the BLOTTO solution (Pierce). Purified phages (˜10¹⁰ pfu)originating from single plaques (M13)/colonies (fUSE5) are added to eachwell and incubated overnight at 4° C. After washing wells with anappropriate buffer (see above), bound phages are detected by thestandard ELISA protocol using anti-M13 Ab (rabbit, Sigma) andanti-rabbit Ig-peroxidase conjugate (Pierce) or using anti-M13Ab-peroxidase conjugate (Pharmacia). Colormetric assays are performedusing TMB (3,3′,5,5′-tetramethylbenzidine, Pierce). The high affinity ofprotein G to immunoglobulins present a special problem; Abs cannot beused in detection. Therefore, to detect anti-protein G FnAbs, fusionphages are immobilized in wells and the binding is then measured usingbiotinylated protein G followed by the detection usingstreptavidin-peroxidase conjugate.

Production of Soluble FnAbs

After preliminary characterization of mutant Fn3s using phage ELISA,mutant genes are subcloned into the expression vector pEW1. Mutantproteins are produced as His•tag fusion proteins and purified, and theirconformation, stability and ligand affinity are characterized.

Thus, Fn3 is the fourth example of a monomeric immunoglobulin-likescaffold that can be used for engineering binding proteins. Successfulselection of novel binding proteins have also been based on minibody,tendamistat and “camelized” immunoglobulin VH domain scaffolds (Martinet al., 1994; Davies & Riechmann, 1995; McConnell & Hoess, 1995). TheFn3 scaffold has advantages over these systems. Bianchi et al. reportedthat the stability of a minibody was 2.5 kcal/mol, significantly lowerthan that of Ubi4-K No detailed structural characterization ofminibodies has been reported to date. Tendamistat and the VH domaincontain disulfide bonds, and thus preparation of correctly foldedproteins may be difficult Davies and Riechmann reported that the yieldsof their camelized VH domains were less than 1 mg per liter culture(Davies & Riechmann, 1996).

Thus, the Fn3 framework can be used as a scaffold for molecularrecognition. Its small size, stability and well-characterized structuremake Fn3 an attractive system In light of the ubiquitous presence of Fn3in a wide variety of natural proteins involved in ligand binding, onecan engineer Fn3-based binding proteins to different classes of targets.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE I Construction of the Fn3 Gene

A synthetic gene for tenth Fn3 of fibronectin (FIG. 1) was designed onthe basis of amino acid residue 1416-1509 of human fibronectin(Kornblihtt, et al., 1985) and its three dimensional structure (Main, etal., 1992). The gene was engineered to include convenient restrictionsites for mutagenesis and the so-called “preferred codons” for highlevel protein expression (Gribskov, et al., 1984) were used. Inaddition, a glutamine residue was inserted after the N-terminalmethionine in order to avoid partial processing of the N-terminalmethionine which often degrades NMR spectra (Smith, et al., 1994).Chemical reagents were of the analytical grade or better and purchasedfrom Sigma Chemical Company and J. T. Baker, unless otherwise noted.Recombinant DNA procedures were performed as described in “MolecularCloning” (Sambrook, et al., 1989), unless otherwise stated. Customoligonucleotides were purchased from Operon Technologies. Restrictionand modification enzymes were from New England Biolabs.

The gene was assembled in the following manner. First, the gene sequence(FIG. 5) was divided into five parts with boundaries at designedrestriction sites: fragment 1, NdeI-PstI (oligonucleotides FN 1F andFN1R (Table 2); fragment 2, PstI-EcoRI (FN2F and FN2R); fragment 3,EcoRI-SalI (FN3F and FN3R); fragment 4, SalI-SacI (FN4F and FN4R);fragment 5, SacI-BamHI (FN5F and FN5R). Second, for each part, a pair ofoligonucleotides which code opposite strands and have complementaryoverlaps of approximately 15 bases was synthesized. Theseoligonucleotides were designated FN1F-FN5R and are shown in Table 2.Third, each pair (e.g., FN1F and FN1R) was annealed and single-strandregions were filled in using the Klenow fragment of DNA polymerase.Fourth, the double stranded oligonucleotide was digested with therelevant restriction enzymes at the termini of the fragment and clonedinto the PBLUESCRIPT SK plasmid (Stratagene) which had been digestedwith the same enzymes as those used for the fragments. The DNA sequenceof the inserted fragment was confirmed by DNA sequencing using anApplied Biosystems DNA sequencer and the dideoxy termination protocolprovided by the manufacturer. Last, steps 24 were repeated to obtain theentire gene.

The gene was also cloned into the pET3a and pET15b (Novagen) vectors(pAS45 and pAS25, respectively). The maps of the plasmids are shown inFIGS. 6 and 7. E. coli BL21 (DE3) (Novagen) containing these vectorsexpressed the Fn3 gene under the control of bacteriophage T7 promotor(Studier, et al., 1990); pAS24 expresses the 96-residue Fn3 proteinonly, while pAS45 expresses Fn3 as a fusion protein with poly-histidinepeptide (His-tag). High level expression of the Fn3 protein and itsderivatives in E. coli was detected as an intense band on SDS-PAGEstained with CBB.

The binding reaction of the monobodies is characterized quantitativelyby means of fluorescence spectroscopy using purified soluble monobodies.

Intrinsic fluorescence is monitored to measure binding reactions. Trpfluorescence (excitation at ˜290 nm, emission at 300 350 nm) and Tyrfluorescence (excitation at ˜260 nm, emission at ˜303 nm) is monitoredas the Fn3-mutant solution (≦100 μM) is titrated with a ligand solution.When a ligand is fluorescent (e.g. fluorescein), fluorescence from theligand may be used. K_(d) of the reaction will be determined by thenonlinear least-squares fitting of the bimolecular binding equation.

If intrinsic fluorescence cannot be used to monitor the bindingreaction, monobodies are labeled with fluorescein-NHS (Pierce) andfluorescence polarization is used to monitor the binding reaction (Burkeet al., 1996).

EXAMPLE II Modifications to Include Restriction Sites in the Fn3 Gene

The restriction sites were incorporated in the synthetic Fn3 genewithout changing the amino acid sequence Fn3. The positions of therestriction sites were chosen so that the gene construction could becompleted without synthesizing long (>60 bases) oligonucleotides and sothat two loop regions could be mutated (including by randomization) bythe cassette mutagenesis method (i.e., swapping a fragment with anothersynthetic fragment containing mutations). In addition, the restrictionsites were chosen so that most sites were unique in the vector for phagedisplay. Unique restriction sites allow one to recombine monobody cloneswhich have been already selected in order to supply a larger sequencespace.

EXAMPLE III

Construction of M13 Phage Display Libraries

A vector for phage display, pAS38 (for its map, see FIG. 8) wasconstructed as follows. The XbaI-BamHI fragment of pET12a encoding thesignal peptide of OmpT was cloned at the 5′ end of the Fn3 gene. TheC-terminal region (from the FN5F and FN5R′ oligonucleotides, see Table2) of the Fn3 gene was replaced with a new fragment consisting of theFN5F and FN5R′ oligonucleotides (Table 2) which introduced a MluI siteand a linker sequence for making a fusion protein with the pIII proteinof bacteriophage M13. A gene fragment coding the C-terminal domain ofM13 pIII was prepared from the wild-type gene III of M13 mp18 using PCR(Corey, et al., 1993) and the fragment was inserted at the 3′ end of theOmpT-Fn3 fusion gene using the MluI and HindIII sites.

Phages were produced and purified using a helper phage, M13K07,according to a standard method (Sambrook, et al., 1989) except thatphage particles were purified by a second polyethylene glycolprecipitation. Successful display of Fn3 on fusion phages was confirmedby ELISA (Harlow & Lane, 1988) using an antibody against fibronectin(Sigma) and a custom anti-FN3 antibody (Cocalico Biologicals, PA, USA).

EXAMPLE IV Libraries Containing Loop Variegations in the AB Loop

A nucleic acid phage display library having variegation in the AB loopis prepared by the following methods. Randomization is achieved by theuse of oligonucleotides containing degenerated nucleotide sequence.Residues to be variegated are identified by examining the X-ray and NMRstructures of Fn3 (Protein Data Bank accession numbers, 1FNA and 1TTF,respectively). Oligonucleotides containing NNK (N and K here denote anequimolar mixture of A, T, G, and C and an equimolar mixture of G and T,respectively) for the variegated residues are synthesized (seeoligonucleotides BC3, FG2, FG3, and FG4 in Table 2 for example). The NNKmixture codes for all twenty amino acids and one termination codon(TAG). TAG, however, is suppressed in the E. coli XL-1 blue.Single-stranded DNAs of pAS38 (and its derivatives) are prepared using astandard protocol (Sambrook, et al., 1989).

Site-directed mutagenesis is performed following published methods (seefor example, Kunkel, 1985) using a MUTA-GENE kit (BioRad). The librariesare constructed by electroporation of E. coli XL-1 Blue electroporationcompetent cells (200 μl; Stratagene) with 1 μg of the plasmid DNA usinga BTX electrocell manipulator ECM 395 1 mm gap cuvette. A portion of thetransformed cells is plated on an LB-agar plate containing ampicillin(100 μg/ml) to determine the transformation efficiency. Typically, 3×10⁸transformants are obtained with 1 μg of DNA, and thus a library contains10⁸ to 10⁹ independent clones. Phagemid particles were prepared asdescribed above.

EXAMPLE V Loop Variegations in the BC, CD, DE, EF or FG Loop

A nucleic acid phage display library having five variegated residues(residues number 26-30) in the BC loop, and one having seven variegatedresidues (residue numbers 78-84) in the FG loop, was prepared using themethods described in Example IV above. Other nucleic acid phage displaylibraries having variegation in the CD, DE or EF loop can be prepared bysimilar methods.

EXAMPLE VI Loop Variegations in the FG and BC Loop

A nucleic acid phage display library having seven variegated residues(residues number 78-84) in the FG loop and five variegated residues(residue number 26-30) in the BC loop was prepared. Variegations in theBC loop were prepared by site-directed mutagenesis (Kunkel, et al.)using the BC3 oligonucleotide described in Table 1. Variegations in theFG loop were introduced using site-directed mutagenesis using the BCloop library as the starting material, thereby resulting in librariescontaining variegations in both BC and FG loops. The oligonucleotide FG2has variegating residues 78-84 and oligonucleotide FG4 has variegatingresidues 77-81 and a deletion of residues 82-84.

A nucleic acid phage display library having five variegated residues(residues 78-84) in the FG loop and a three residue deletion (residues82-84) in the FG loop, and five variegated residues (residues 26-30) inthe BC loop, was prepared. The shorter FG loop was made in an attempt toreduce the flexibility of the FG loop; the loop was shown to be highlyflexible in Fn3 by the NMR studies of Main, et al. (1992). A highlyflexible loop may be disadvantageous to forming a binding site with ahigh affinity (a large entropy loss is expected upon the ligand binding,because the flexible loop should become more rigid). In addition, otherFn3 domains (besides human) have shorter FG loops (for sequencealignment, see FIG. 12 in Dickinson, et al. (1994)).

Randomization was achieved by the use of oligonucleotides containingdegenerate nucleotide sequence (oligonucleotide BC3 for variegating theBC loop and oligonucleotides FG2 and FG4 for variegating the FG loops).

Site-directed mutagenesis was performed following published methods (seefor example, Kunkel, 1985). The libraries were constructed byelectrotransforming E. coli XL-1 Blue (Stratagene). Typically a librarycontains 108 to 109 independent clones. Library 2 contains fivevariegated residues in the BC loop and seven variegated residues in theFG loop. Library 4 contains five variegated residues in each of the BCand FG loops, and the length of the FG loop was shortened by threeresidues.

EXAMPLE VII fd Phage Display Libraries Constructed with LoopVariegations

Phage display libraries are constructed using the fd phage as thegenetic vector. The Fn3 gene is inserted in fUSE5 (Parmley & Smith,1988) using SFlI restriction sites which are introduced at the 5′ and 3′ends of the Fn3 gene using PCR. The expression of this phage results inthe display of the fusion pIII protein on the surface of the fd phage.Variegations in the Fn3 loops are introduced using site-directedmutagenesis as described hereinabove, or by subcloning the Fn3 librariesconstructed in M13 phage into the fUSE5 vector.

EXAMPLE VIII Other Phage Display Libraries

T7 phage libraries (Novagen, Madison, Wis.) and bacterial piliexpression systems (Invitrogen) are also useful to express the Fn3 gene.

EXAMPLE IX Isolation of Polypeptides which Bind to MacromolecularStructures

The selection of phage-displayed monobodies was performed following theprotocols of Barbas and coworkers (Rosenblum & Barbas, 1995). Briefly,approximately 1 μg of a target molecule (“antigen”) in sodium carbonatebuffer (100 mM, pH 8.5) was immobilized in the wells of a microtiterplate (Maxisorp, Nunc) by incubating overnight at 4° C. in an air tightcontainer. After the removal of this solution, the wells were thenblocked with a 3% solution of BSA (Sigma, Fraction V) in TBS byincubating the plate at 37° C. for 1 hour. A phagemid library solution(50 μl) containing approximately 10¹² colony forming units (cfu) ofphagemid was absorbed in each well at 37° C. for 1 hour. The wells werethen washed with an appropriate buffer (typically TBST, 50 mM Tris-HCl(pH 7.5), 150 mM NaCl, and 0.5% Tween20) three times (once for the firstround). Bound phage were eluted by an acidic solution (typically, 0.1 Mglycine-HCl, pH 2.2; 50 μl) and recovered phage were immediatelyneutralized with 3 μl of Tris solution. Alternatively, bound phage wereeluted by incubating the wells with 50 μl of TBS containing the antigen(1-10 μM). Recovered phage were amplified using the standard protocolemploying the XL1Blue cells as the host (Sambrook, et al.). Theselection process was repeated 5-6 times to concentrate positive clones.After the final round, individual clones were picked and their bindingaffinities and DNA sequences were determined.

The binding affinities of monobodies on the phage surface werecharacterized using the phage ELISA technique (Li, et al., 1995). Wellsof microtiter plates (Nunc) were coated with an antigen and blocked withBSA. Purified phages (10⁸-10¹¹ cfu) originating from a single colonywere added to each well and incubated 2 hours at 37° C. After washingwells with an appropriate buffer (see above), bound phage were detectedby the standard ELISA protocol using antiM13 antibody (rabbit, Sigma)and anti-rabbit Ig-peroxidase conjugate (Pierce). Colorimetric assayswere performed using Turbo-TMB (3,3′,5,5′-tetramethylbenzidine, Pierce)as a substrate.

The binding affinities of monobodies on the phage surface were furthercharacterized using the competition ELISA method (Djavadi-Ohaniance, etal., 1996). In this experiment, phage ELISA is performed in the samemanner as described above, except that the phage solution contains aligand at varied concentrations. The phage solution was incubated a 4°C. for one hour prior to the binding of an immobilized ligand in amicrotiter plate well. The affinities of phage displayed monobodies areestimated by the decrease in ELISA signal as the free ligandconcentration is increased.

After preliminary characterization of monobodies displayed on thesurface of phage using phage ELISA, genes for positive clones weresubcloned into the expression vector pAS45. E. coli BL21(DE3) (Novagen)was transformed with an expression vector (pAS45 and its derivatives).Cells were grown in M9 minimal medium and M9 medium supplemented withBactotryptone (Difco) containing ampicillin (200 μg/1 ml). For isotopiclabeling, ¹⁵N NH⁴Cl and/or ¹³C glucose replaced unlabeled components.Stable isotopes were purchased from Isotec and Cambridge Isotope Labs.500 ml medium in a 2 1 baffle flask was inoculated with 10 ml ofovernight culture and agitated at approximately 140 rpm at 37° C. IPTGwas added at a final concentration of 1 mM to induce protein expressionwhen OD(600 nm) reached approximately 1.0. The cells were harvested bycentrifugation 3 hours after the addition of IPTG and kept frozen at−70° C. until used.

Fn3 and monobodies with His•tag were purified as follows. Cells weresuspended in 5 ml/(g cell) of 50 mM Tris (pH 7.6) containing 1 mMphenylmethylsulfonyl fluoride. HEL (Sigma, 3X crystallized) was added toa final concentration of 0.5 mg/mL After incubating the solution for 30min at 37° C., it was sonicated so as to cause cell breakage three timesfor 30 seconds on ice. Cell debris was removed by centrifugation at15,000 rpm in an Sorval RC-2B centrifuge using an SS-34 rotor.Concentrated sodium chloride is added to the solution to a finalconcentration of 0.5 M. The solution was then applied to a 1 ml HISTRAP™chelating column (Pharmacia) preloaded with nickel chloride (0.1 M, 1ml) and equilibrated in the Tris buffer (50 mM, pH 8.0) containing 0.5 Msodium chloride. After washing the column with the buffer, the boundprotein was eluted with a Tris buffer (50 mM, pH 8.0) containing 0.5 Mimidazole. The His•tag portion was cleaved off, when required, bytreating the fusion protein with thrombin using the protocol supplied byNovagen (Madison, Wis.). Fn3 was separated from the His-tag peptide andthrombin by a RESOURCES® column (Pharmacia) using a linear gradient ofsodium chloride (0-0.5 M) in sodium acetate buffer (20 mM, pH 5.0).

Small amounts of soluble monobodies were prepared as follows. XL-1 Bluecells containing pAS38 derivatives (plasmids coding Fn3-pIII fusionproteins) were grown in LB media at 37° C. with vigorous shaking untilOD(600 nm) reached approximately 1.0; IPTG was added to the culture to afinal concentration of 1 mM, and the cells were further grown overnightat 37° C. Cells were removed from the medium by centrifugation, and thesupernatant was applied to a microtiter well coated with a ligand.Although XL-1 Blue cells containing pAS38 and its derivatives expressFN3-pIII fusion proteins, soluble proteins are also produced due to thecleavage of the linker between the Fn3 and pIII regions by proteolyticactivities of E. coli (Rosenblum & Barbas, 1995). Binding of a monobodyto the ligand was examined by the standard ELISA protocol using a customantibody against Fn3 (purchased from Cocalico Biologicals, Reamstown,Pa.). Soluble monobodies obtained from the periplasmic fraction of E.coli cells using a standard osmotic shock method were also used.

EXAMPLE X Ubiquitin Binding Monobody

Ubiquitin is a small (76 residue) protein involved in the degradationpathway in eurkaryotes. It is a single domain globular protein. Yeastubiquitin was purchased from Sigma Chemical Company and was used withoutfurther purification.

Libraries 2 and 4, described in Example VI above, were used to selectubiquitin-binding monobodies. Ubiquitin (1 μg in 50 μl sodiumbicarbonate buffer (100 mM, pH 8.5)) was immobilized in the wells of amicrotiter plate, followed by blocking with BSA (3% in TBS). Panning wasperformed as described above. In the first two rounds, 1 μg of ubiquitinwas immobilized per well, and bound phage were elute with an acidicsolution. From the third to the sixth rounds, 0.1 μg of ubiquitin wasimmobilized per well and the phage were eluted either with an acidicsolution or with TBS containing 10 μM ubiquitin.

Binding of selected clones was tested first in the polyclonal mode,i.e., before isolating individual clones. Selected clones from alllibraries showed significant binding to ubiquitin. These results areshown in FIG. 9. The binding to the immobilized ubiquitin of the cloneswas inhibited almost completely by less than 30 μm soluble ubiquitin inthe competition ELISA experiments (see FIG. 10). The sequences of the BCand FG loops of ubiquitin-binding monobodies is shown in Table 3. TABLE3 Sequences of ubiquitin-binding monobodies Occurrence (if more Name BCloop FG loop than one) 211 CARRA (SEQ ID NO:31) RWIPLAK (SEQ ID NO:32) 2212 CWRRA (SEQ ID NO:33) RWVGLAW (SEQ ID NO:34) 213 CKHRR (SEQ ID NO:35)FADLWWR (SEQ ID NO:36) 214 CRRGR (SEQ ID NO:37) RGFMWLS (SEQ ID NO:38)215 CNWRR (SEQ ID NO:39) RAYRYRW (SEQ ID NO:40) 411 SRLRR (SEQ ID NO:41)PPWRV (SEQ ID NO:42) 9 422 ARWTL (SEQ ID NO:43) RRWWW (SEQ ID NO:44) 424GQRTF (SEQ ID NO:45) RRWWA (SEQ ID NO:46)

The 411 clone, which was the most enriched clone, was characterizedusing phage ELISA. The 411 clone showed selective binding and inhibitionof binding in the presence of about 10 μM ubiquitin in solution (FIG.11).

EXAMPLE XI Methods for the Immobilization of Small Molecules

Target molecules were immobilized in wells of a microtiter plate(MAXISORP, Nunc) as described hereinbelow, and the wells were blockedwith BSA.

In addition to the use of carrier protein as described below, aconjugate of a target molecule in biotin can be made. The biotinylatedligand can then be immobilized to a microtiter plate well which has beencoated with streptavidin.

In addition to the use of a carrier protein as described below, onecould make a conjugate of a target molecule and biotin (Pierce) andimmobilize a biotinylated ligand to a microtiter plate well which hasbeen coated with streptavidin (Smith and Scott, 1993).

Small molecules may be conjugated with a carrier protein such as bovineserum albumin (BSA, Sigma), and passively adsorbed to the microtiterplate well Alternatively, methods of chemical conjugation can also beused. In addition, solid supports other than microtiter plates canreadily be employed.

EXAMPLE XII Fluorescein Binding Monobody

Fluorescein has been used as a target for the selection of antibodiesfrom combinatorial libraries (Barbas, et al. 1992). NHS-fluorescein wasobtained from Pierce and used according to the manufacturer'sinstructions in preparing conjugates with BSA (Sigma). Two types offluorescein-BSA conjugates were prepared with approximate molar ratiosof 17 (fluorescein) to one (BSA).

The selection process was repeated 5-6 times to concentrate positiveclones. In this experiment, the phage library was incubated with aprotein mixture (BSA, cytochrome C (Sigma, Horse) and RNaseA (Sigma,Bovine), 1 mg/ml each) at room temperature for 30 minutes, prior to theaddition to ligand coated wells. Bound phage were eluted in TBScontaining 10 μM soluble fluorescein, instead of acid elution After thefinal round, individual clones were picked and their binding affinities(see below) and DNA sequences were determined. TABLE 4 BC FG Clones fromLibrary #2 WT AVTVR RGDSPAS (SEQ ID NO:47) (SEQ ID NO:48) pLB24.1 CNWRRRAYRYRW (SEQ ID NO:49) (SEQ ID NO:50) pLB24.2 CMWRA RWGMLRR (SEQ IDNO:51) (SEQ ID NO:52) pLB24.3 ARMRE RWLRGRY (SEQ ID NO:53) (SEQ IDNO:54) pLB24.4 CARRR RRAGWGW (SEQ ID NO:55) (SEQ ID NO:56) pLB24.5 CNWRRRAYRYRW (SEQ ID NO:57) (SEQ ID NO:58) pLB24.6 RWRER RHPWTER (SEQ IDNO:59) (SEQ ID NO:60) pLB24.7 CNWRR RAYRYRW (SEQ ID NO:61) (SEQ IDNO:62) pLB24.8 ERRVP RLLLWQR (SEQ ID NO:63) (SEQ ID NO:64) pLB24.9 GRGAGFGSFERR (SEQ ID NO:65) (SEQ ID NO:66) pLB24.11 CRWTR RRWFDGA (SEQ IDNO:67) (SEQ ID NO:68) pLB24.12 CNWRR RAYRYRW (SEQ ID NO:69) (SEQ IDNO:70) Clones from Library #4 WT AVTVR GRGDS (SEQ ID NO:71) (SEQ IDNO:72) pLB25.1 GQRTF RRWWA (SEQ ID NO:73) (SEQ ID NO:74) pLB25.2 GQRTFRRWWA (SEQ ID NO:75) (SEQ ID NO:76) pLB25.3 GQRTF RRWWA (SEQ ID NO:77)(SEQ ID NO:78) pLB25.4 LRYRS GWRWR (SEQ ID NO:79) (SEQ ID NO:80) pLB25.5GQRTF RRWWA (SEQ ID NO:81) (SEQ ID NO:82) pLB25.6 GQRTF RRWWA (SEQ IDNO:83) (SEQ ID NO:84) pLB25.7 LRYRS GWRWR (SEQ ID NO:85) (SEQ ID NO:86)pLB25.9 LRYRS GWRWR (SEQ ID NO:87) (SEQ ID NO:88) pLB25.11 GQRTF RRWWA(SEQ ID NO:89) (SEQ ID NO:90) pLB25.12 LRYRS GWRWR (SEQ ID NO:91) (SEQID NO:92)

Preliminary characterization of the binding affinities of selectedclones were performed using phage ELISA and competition phage ELISA (seeFIG. 12 (Fluorescein-1) and FIG. 13 (Fluorescein-2)). The four clonestested showed specific binding to the ligand-coated wells, and thebinding reactions are inhibited by soluble fluorescein (see FIG. 13).

EXAMPLE XIII Digoxigenin Binding Monobody

Digoxigenin-3-O-methyl-carbonyl-e-aminocapronic acid-NHS (BoehringerMannheim) is used to prepare a digoxigenin-BSA conjugate. The couplingreaction is performed following the manufacturers' instructions. Thedigoxigenin-BSA conjugate is immobilized in the wells of a microtiterplate and used for panning. Panning is repeated 5 to 6 times to enrichbinding clones. Because digoxigenin is sparingly soluble in aqueoussolution, bound phages are eluted from the well using acidic solution.See Example XIV.

EXAMPLE XIV TSAC (Transition State Analog Compound) Binding Monobodies

Carbonate hydrolyzing monobodies are selected as follows. A transitionstate analog for carbonate hydrolysis, 4-nitrophenyl phosphonate issynthesized by an Arbuzov reaction as described previously (Jacobs andSchultz, 1987). The phosphonate is then coupled to the carrier protein,BSA, using carbodiimide, followed by exhaustive dialysis (Jacobs andSchultz, 1987). The hapten-BSA conjugate is immobilized in the wells ofa microtiter plate and monobody selection is performed as describedabove. Catalytic activities of selected monobodies are tested using4-nitrophenyl carbonate as the substrate.

Other haptens useful to produce catalytic monobodies are summarized inH. Suzuki (1994) and in N. R Thomas (1994).

EXAMPLE XV NMR Characterization of Fn3 and Comparison of the Fn3Secreted by Yeast With That Secreted by E. coli

Nuclear magnetic resonance (NMR) experiments are performed to identifythe contact surface between FnAb and a target molecule, e.g., monobodiesto fluorescein, ubiquitin, RNaseA and soluble derivatives ofdigoxigenin. The information is then be used to improve the affinity andspecificity of the monobody. Purified monobody samples are dissolved inan appropriate buffer for NMR spectroscopy using Amicon ultrafiltrationcell with a YM-3 membrane. Buffers are made with 90% H₂O/10% D₂O(distilled grade, Isotec) or with 100% D₂O. Deuterated compounds (e.g.acetate) are used to eliminate strong signals from them.

NMR experiments are performed on a Varian Unity INOVA 600 spectrometerequipped with four RF channels and a triple resonance probe with pulsedfield gradient capability. NMR spectra are analyzed using processingprograms such as FELIX (Molecular Simulations), NMRPIPE, PIPP, and CAPP(Garrett, et al., 1991; Delaglio, et al., 1995) on UNIX workstations.Sequence specific resonance assignments are made using well-establishedstrategy using a set of triple resonance experiments (CBCA(CO)NH andHNCACB) (Grzesiek & Bax, 1992; Wittenkind & Mueller, 1993).

Nuclear Overhauser effect (NOE) is observed between ¹H nuclei closerthan approximately 5 Å, which allows one to obtain information oninterproton distances. A series of double- and triple-resonanceexperiments (Table 5; for recent reviews on these techniques, see Bax &Grzesiek, 1993 and Kay, 1995) are performed to collect distance (i.e.NOE) and dihedral angle (J-coupling) constraints. Isotope-filteredexperiments are performed to determine resonance assignments of thebound ligand and to obtain distance constraints within the ligand andthose between FnAb and the ligand. Details of sequence specificresonance assignments and NOE peak assignments have been described indetail elsewhere (Clore & Gronenborn, 1991; Pascal, et al., 1994b;Metzler, et al., 1996). TABLE 5 NMR experiments for structurecharacterization Experiment Name Reference 1. reference spectra 2D-¹H,¹⁵N-HSQC (Bodenhausen & Ruben, 1980; Kay, et al., 1992) 2D-¹H, ¹³C-HSQC(Bodenhausen & Ruben, 1980; Vuister & Bax, 1992) 2. backbone and sidechain resonance assignments of ¹³C/¹⁵N-labeled protein 3D-CBCA(CO)NH(Grzesiek & Bax, 1992) 3D-HNCACB (Wittenkind & Mueller, 1993) 3D-C(CO)NH(Logan et al., 1992; Grzesiek et al., 1993) 3D-H(CCO)NH3D-HBHA(CBCACO)NH (Grzesiek & Bax, 1993) 3D-HCCH-TOCSY (Kay et al.,1993) 3D-HCCH-COSY (Ikura et al., 1991) 3D-¹H, ¹⁵N-TOCSY-HSQC (Zhang etal., 1994) 2D-HB(CBCDCE)HE (Yamazaki et al., 1993) 3. resonanceassignments of unlabeled ligand 2D-isotope-filtered ¹H-TOCSY2D-isotope-filtered ¹H-COSY 2D-isotope-filtered ¹H-NOESY (Ikura & Bax,1992) 4. structural constraints within labeled protein 3D-¹H,¹⁵N-NOESY-HSQC (Zhang et al., 1994) 4D-¹H, ¹³C-HMQC-NOESY-HMQC (Vuisteret al., 1993) 4D-¹H, ¹³C, ¹⁵N-HSQC-NOESY-HSQC (Muhandiram et al., 1993;Pascal et al., 1994a) within unlabeled ligand 2D-isotope-filtered¹H-NOESY (Ikura & Bax, 1992) interactions between protein and ligand3D-isotope-filtered ¹H, ¹⁵N-NOESY-HSQC 3D-isotope-filtered ¹H, (Lee etal., 1994) ¹³C-NOESY-HSQC 5. dihedral angle constraints J-molulated¹H,¹⁵N-HSQC (Billeter et al., 1992) 3D-HNHB (Archer et al., 1991)

Backbone ¹H, ¹⁵N and ¹³C resonance assignments for a monobody arecompared to those for wild-type Fn3 to assess structural changes in themutant Once these data establish that the mutant retains the globalstructure, structural refinement is performed using experimental NOEdata. Because the structural difference of a monobody is expected to beminor, the wild-type structure can be used as the initial model aftermodifying the amino acid sequence. The mutations are introduced to thewild-type structure by interactive molecular modeling, and then thestructure is energy-minimized using a molecular modeling program such asQUANTA (Molecular Simulations). Solution structure is refined usingcycles of dynamical simulated annealing (Nilges et al., 1988) in theprogram X-PLOR (Brünger, 1992). Typically, an ensemble of fiftystructures is calculated. The validity of the refined structures isconfirmed by calculating a fewer number of structures from randomlygenerated initial structures in X-PLOR using the YASAP protocol (Nilges,et al., 1991). Structure of a monobody-ligand complex is calculated byfirst refining both components individually using intramolecular NOEs,and then docking the two using intermolecular NOEs.

For example, the ¹H, ¹⁵N-HSQC spectrum for the fluorescein-bindingmonobody LB25.5 is shown in FIG. 14. The spectrum shows a gooddispersion (peaks are spread out) indicating that LB25.5 is folded intoa globular conformation. Further, the spectrum resembles that for thewild-type Fn3, showing that the overall structure of LB25.5 is similarto that of Fn3. These results demonstrate that ligand-binding monobodiescan be obtained without changing the global fold of the Fn3 scaffold.

Chemical shift perturbation experiments are performed by forming thecomplex between an isotope-labeled FnAb and an unlabeled ligand. Theformation of a stoichiometric complex is followed by recording the HSQCspectrum. Because chemical shift is extremely sensitive to nuclearenvironment, formation of a complex usually results in substantialchemical shift changes for resonances of amino acid residues in theinterface. Isotope-edited NMR experiments (2D HSQC and 3D CBCA(CO)NH)are used to identify the resonances that are perturbed in the labeledcomponent of the complex; i.e. the monobody. Although the possibility ofartifacts due to long-range conformational changes must always beconsidered, substantial differences for residues clustered on continuoussurfaces are most likely to arise from direct contacts (Chen et al.,1993; Gronenborn & Clore, 1993).

An alternative method for mapping the interaction surface utilizes amidehydrogen exchange (HX) measurements. HX rates for each amide proton aremeasured for ¹⁵N labeled monobody both free and complexed with a ligand.Ligand binding is expected to result in decreased amide HX rates formonobody residues in the interface between the two proteins, thusidentifying the binding surface. HX rates for monobodies in the complexare measured by allowing HX to occur for a variable time followingtransfer of the complex to D₂O; the complex is dissociated by loweringpH and the HSQC spectrum is recorded at low pH where amide HX is slow.Fn3 is stable and soluble at low pH, satisfying the prerequisite for theexperiments.

EXAMPLE XVI Construction and Analysis of Fn3-Display System Specific forUbiquitin

An Fn3-display system was designed and synthesized, ubiquitin-bindingclones were isolated and a major Fn3 mutant in these clones wasbiophysically characterized.

Gene construction and phage display of Fn3 was performed as in ExamplesI and II above. The Fn3-phage pIII fusion protein was expressed from aphagemid-display vector, while the other components of the M13 phage,including the wildtype pIII, were produced using a helper phage (Bass etal., 1990). Thus, a phage produced by this system should contain lessthan one copy of Fn3 displayed on the surface. The surface display ofFn3 on the phage was detected by ELISA using an anti-Fn3 antibody. Onlyphages containing the Fn3-pIII fusion vector reacted with the antibody.

After confirming the phage surface to display Fn3, a phage displaylibrary of Fn3 was constructed as in Example III. Random sequences wereintroduced in the BC and FG loops. In the first library, five residues(77-81) were randomized and three residues (82-84) were deleted from theFG loop. The deletion was intended to reduce the flexibility and improvethe binding affinity of the FG loop. Five residues (26-30) were alsorandomized in the BC loop in order to provide a larger contact surfacewith the target molecule. Thus, the resulting library contains fiverandomized residues in each of the BC and FG loops (Table 6). Thislibrary contained approximately 10⁸ independent clones.

Library Screening

Library screening was performed using ubiquitin as the target molecule.In each round of panning, Fn3-phages were absorbed to a ubiquitin-coatedsurface, and bound phages were eluted competitively with solubleubiquitin. The recovery ratio improved from 4.3×10⁻⁷ in the second roundto 4.5×10⁻⁶ in the fifth round, suggesting an enrichment of bindingclones. After five founds of panning, the amino acid sequences ofindividual clones were determined (Table 6). TABLE 6 Sequences in thevariegated loops of enriched clones Name BC loop FG loop Frequency WildType GCAGTTACCGTGCGT GGCCGTGGTGAGAGCCCAGCGAGC — (SEQ ID NO:93) (SEQ IDNO:95) AlaValThrValArg GlyArgGlyAspSerProAlaSer (SEQ ID NO:94) (SEQ IDNO:96) Library^(a) NNKNNKNNKNNKNNK NNKNNKNNKNNKNNK--------- — XXXXXXXXXX (deletion) clone 1 TCGAGGTTGCGGCGG CCGCCGTGGAGGGTG 9 (SEQ IDNO:97) (SEQ ID NO:99) (Ubi4) SerArgLeuArgArg ProProTrpArgVal (SEQ IDNO:98) (SEQ ID NO:100) clone2 GGTCAGCGAACTTTT AGGCGGTGGTGGGCT 1 (SEQ IDNO:101) (SEQ ID NO:103) GlyGlnArgThrPhe ArgArgTrpTrpAla (SEQ ID NO:102)(SEQ ID NO:104) clone3 GCGAGGTGGACGCTT AGGCGGTGGTGGTGG 1 (SEQ ID NO:105)(SEQ ID NO:107) AlaArgTrpThrLeu ArgArgTrpTrpTrp (SEQ ID NO:106) (SEQ IDNO:108)^(a)N denotes an equimolar mixture of A, T, G and C; K denotes anequimolar mixture of G and T.A clone, dubbed Ubi4, dominated the enriched pool of Fn3 variants.Therefore, further investigation was focused on this Ubi4 clone. Ubi4contains four mutations in the BC loop (Arg 30 in the BC loop wasconserved) and five mutations and three deletions in the FG loop. Thus13% (12 out of 94) of the residues were altered in Ubi4 from thewild-type sequence.

FIG. 15 shows a phage ELISA analysis of Ubi4. The Ubi4 phage binds tothe target molecule, ubiquitin, with a significant affinity, while aphage displaying the wild-type Fn3 domain or a phase with no displayedmolecules show little detectable binding to ubiquitin (FIG. 15 a). Inaddition, the Ubi4 phage showed a somewhat elevated level of backgroundbinding to the control surface lacking the ubiquitin coating. Acompetition ELISA experiments shows the IC₅₀ (concentration of the freeligand which causes 50% inhibition of binding) of the binding reactionis approximately 5 μM (FIG. 15 b). BSA, bovine ribonuclease A andcytochrome C show little inhibition of the Ubi4-ubiquitin bindingreaction (FIG. 15 c), indicating that the binding reaction of Ubi4 toubiquitin does result from specific binding.

Characterization of a Mutant Fn3 Protein

The expression system yielded 50-100 mg Fn3 protein per liter culture. Asimilar level of protein expression was observed for the Ubi4 clone andother mutant Fn3 proteins.

Ubi4-Fn3 was expressed as an independent protein. Though a majority ofUbi4 was expressed in E. coli as a soluble protein, its solubility wasfound to be significantly reduced as compared to that of wild-type Fn3.Ubi4 was soluble up to ˜20 μM at low pH, with much lower solubility atneutral pH This solubility was not high enough for detailed structuralcharacterization using NMR spectroscopy or X-ray crystallography.

The solubility of the Ubi4 protein was improved by adding a solubilitytail, GKKGK, as a C-terminal extension The gene for Ubi4-Fn3 wassubcloned into the expression vector pAS45 using PCR. The C-terminalsolubilization tag, GKKGK, was incorporated in this step. E. coli BL21(DE3) (Novagen) was transformed with the expression vector (pAS45 andits derivatives). Cells were grown in M9 minimal media and M9 mediasupplemented with Bactotryptone (Difco) containing ampicillin (200μg/ml). For isotopic labeling, ¹⁵N NH₄Cl replaced unlabeled NH₄Cl in themedia 500 ml medium in a 2 liter baffle flask was inoculated with 10 mlof overnight culture and agitated at 37° C. IPTG was added at a finalconcentration of 1 mM to initiate protein expression when OD (600 nm)reaches one. The cells were harvested by centrifugation 3 hours afterthe addition of IPTG and kept frozen at 70° C. until used.

Proteins were purified as follows. Cells were suspended in 5 ml/(g cell)of Tris (50 mM, pH 7.6) containing phenylmethylsulfonyl fluoride (1 mM).Hen egg lysozyme (Sigma) was added to a final concentration of 0.5 mg/mLAfter incubating the solution for 30 minutes at 37° C., it was sonicatedthree times for 30 seconds on ice. Cell debris was removed bycentrifugation Concentrated sodium chloride was added to the solution toa final concentration of 0.5 M. The solution was applied to a HI-TRAPchelating column (Pharmacia) preloaded with nickel and equilibrated inthe Tris buffer containing sodium chloride (0.5 M). After washing thecolumn with the buffer, histag-Fn3 was eluted with the buffer containing500 mM imidazole. The protein was further purified using a RESOURCES®column (Pharmacia) with a NaCl gradient in a sodium acetate buffer (20mM, pH 4.6).

With the GKKGK (SEQ ID NO: 109) tail, the solubility of the Ubi4 proteinwas increased to over 1 mM at low pH and up to ˜50 μM at neutral pH.Therefore, further analyses were performed on Ubi4 with this C-terminalextension (hereafter referred to as Ubi4-K). It has been reported thatthe solubility of a minibody could be significantly improved by additionof three Lys residues at the N- or C-termini (Bianchi et al., 1994). Inthe case of protein Rop, a non-structured C-terminal tail is critical inmaintaining its solubility (Smith et al., 1995).

Oligomerization states of the Ubi4 protein were determined using a sizeexclusion column. The wild-type Fn3 protein was monomeric at low andneutral pH's However, the peak of the Ubi4-K protein was significantlybroader than that of wild-type Fn3, and eluted after the wild-typeprotein. This suggests interactions between Ubi4-K and the columnmaterial, precluding the use of size exclusion chromatography todetermine the oligomerization state of Ubi4. NMR studies suggest thatthe protein is monomeric at low pH.

The Ubi4-K protein retained a binding affinity to ubiquitin as judged byELISA (FIG. 15 d). However, an attempt to determine the dissociationconstant using a biosensor (Affinity Sensors, Cambridge, U.K.) failedbecause of high background binding of Ubi4-K-Fn3 to the sensor matrix.This matrix mainly consists of dextran, consistent with our observationthat interactions between Ubi4-K interacts with the cross-linked dextranof the size exclusion column.

EXAMPLE XVII Stability Measurements of Monobodies

Guanidine hydrochloride (GuHCl)-induced unfolding and refoldingreactions were followed by measuring tryptophan fluorescence.Experiments were performed on a Spectronic AB-2 spectrofluorometerequipped with a motordriven syringe (Hamilton Co.). The cuvettetemperature was kept at 30° C. The spectrofluorometer and the syringewere controlled by a single computer using a home-built interface. Thissystem automatically records a series of spectra following GuHCltitration. An experiment started with a 1.5 ml buffer solutioncontaining 5 μM protein. An emission spectrum (300400 nm; excitation at290 nm) was recorded following a delay (3-5 minutes) after eachinjection (50 or 100 μl) of a buffer solution containing GUHCl. Thesesteps were repeated until the solution volume reached the full capacityof a cuvette (3.0 ml). Fluorescence intensities were normalized asratios to the intensity at an isofluorescent point which was determinedin separate experiments. Unfolding curves were fitted with a two-statemodel using a nonlinear least-squares routine (Santoro & Bolen, 1988).No significant differences were observed between experiments with delaytimes (between an injection and the start of spectrum acquisition) of 2minutes and 10 minutes, indicating that the unfolding/refoldingreactions reached close to an equilibrium at each concentration pointwithin the delay times used.

Conformational stability of Ubi4-K was measured using above-describedGuHCl-induced unfolding method. The measurements were performed undertwo sets of conditions; first at pH 3.3 in the presence of 300 mM sodiumchloride, where Ubi4-K is highly soluble, and second in TBS, which wasused for library screening. Under both conditions, the unfoldingreaction was reversible, and we detected no signs of aggregation orirreversible unfolding. FIG. 16 shows unfolding transitions of Ubi4-Kand wild-type Fn3 with the N-terminal (hiS)₆ tag and the C-terminalsolubility tag. The stability of wild-type Fn3 was not significantlyaffected by the addition of these tags. Parameters characterizing theunfolding transitions are listed in Table 7. TABLE 7 Stabilityparameters for Ubi4 and wild-type Fn3 as determined by GuHCl-inducedunfolding Protein ΔG₀ (kcal mol⁻¹) m_(G) (kcal mol⁻¹ M⁻¹) Ubi4 (pH 7.5)4.8 ± 0.1 2.12 ± 0.04 Ubi4 (pH 3.3) 6.5 ± 0.1 2.07 ± 0.02 Wild-type (pH7.5) 7.2 ± 0.2 1.60 ± 0.04 Wild-type (pH 3.3) 11.2 ± 0.1  2.03 ± 0.02

ΔG₀ is the free energy of unfolding in the absence of denaturant; m_(G)is the dependence of the free energy of unfolding on GuHClconcentration. For solution conditions, see FIG. 4 caption.

Though the introduced mutations in the two loops certainly decreased thestability of Ubi4-K relative to wild-type Fn3, the stability of Ubi4remains comparable to that of a “typical” globular protein. It shouldalso be noted that the stabilities of the wild-type and Ubi4-K proteinswere higher at pH 3.3 than at pH 7.5.

The Ubi4 protein had a significantly reduced solubility as compared tothat of wild-type Fn3, but the solubility was improved by the additionof a solubility tail. Since the two mutated loops comprise the onlydifferences between the wild-type and Ubi4 proteins, these loops must bethe origin of the reduced solubility. At this point, it is not clearwhether the aggregation of Ubi4-K is caused by interactions between theloops, or by interactions between the loops and the invariable regionsof the Fn3 scaffold.

The Ubi4-K protein retained the global fold of Fn3, showing that thisscaffold can accommodate a large number of mutations in the two loopstested. Though the stability of the Ubi4-K protein is significantlylower than that of the wild-type Fn3 protein, the Ubi4 protein still hasa conformational stability comparable to those for small globularproteins. The use of a highly stable domain as a scaffold is clearlyadvantageous for introducing mutations without affecting the global foldof the scaffold. In addition, the GuHCl-induced unfolding of the Ubi4protein is almost completely reversible. This allows the preparation ofa correctly folded protein even when a Fn3 mutant is expressed in amisfolded form, as in inclusion bodies. The modest stability of Ubi4 inthe conditions used for library screening indicates that Fn3 variantsare folded on the phage surface. This suggests that a Fn3 clone isselected by its binding affinity in the folded form, not in a denaturedform Dickinson et al. proposed that Val 29 and Arg 30 in the BC loopstabilize Fn3. Val 29 makes contact with the hydrophobic core, and Arg30 forms hydrogen bonds with Gly 52 and Val 75. In Ubi4-Fn3, Val 29 isreplaced with Arg, while Arg 30 is conserved. The FG loop was alsomutated in the library. This loop is flexible in the wild-typestructure, and shows a large variation in length among human Fn3 domains(Main et al., 1992). These observations suggest that mutations in the FGloop may have less impact on stability. In addition, the N-terminal tailof Fn3 is adjacent to the molecular surface formed by the BC and FGloops (FIGS. 1 and 17) and does not form a well-defined structure.Mutations in the N-terminal tail would not be expected to have strongdetrimental effects on stability. Thus, residues in the N-terminal tailmay be good sites for introducing additional mutations.

EXAMPLE XVIII NMR SPECTROSCOPY of Ubi4Fn3

Ubi4-Fn3 was dissolved in [²H]-Gly HCl buffer (20 mM, pH 3.3) containingNaCl (300 mM) using an Amicon ultrafiltration unit The final proteinconcentration was 1 mM. NMR experiments were performed on a Varian UnityINOVA 600 spectrometer equipped with a triple-resonance probe withpulsed field gradient The probe temperature was set at 30° C. HSQC,TOCSY-HSQC and NOESY-HSQC spectra were recorded using publishedprocedures (Kay et al., 1992; Zhang et al., 1994). NMR spectra wereprocessed and analyzed using the NMRPIPE and NMRVIEW software (Johnson &Blevins, 1994; Delaglio et al., 1995) on UNIX workstations.Sequence-specific resonance assignments were made using standardprocedures (Wuthrich, 1986; Clore & Gronenborn, 1991). The assignmentsfor wild-type Fn3 (Baron et al., 1992) were confirmed using a¹⁵N-labeled protein dissolved in sodium acetate buffer (50 mM, pH 4.6)at 30° C.

The three-dimensional structure of Ubi4-K was characterized using thisheteronuclear NMR spectroscopy method A high quality spectrum could becollected on a 1 mM solution of ⁵N-labeled Ubi4 (FIG. 17 a) at low pHThe linewidth of amide peaks of Ubi4-K was similar to that of wild-typeFn3, suggesting that Ubi4-K is monomeric under the conditions used.Complete assignments for backbone “H and ”⁵N nuclei were achieved usingstandard ¹H, ¹⁵N double resonance techniques, except for a row of Hisresidues in the N-terminal (His)₆ tag There were a few weak peaks in theHSQC spectrum which appeared to originate from a minor speciescontaining the N-terminal Met residue. Mass spectroscopy analysis showedthat a majority of Ubi4-K does not contain the N-terminal Met residue.FIG. 17 shows differences in ¹HN and ¹⁵N chemical shifts between Ubi4-Kand wild-type Fn3. Only small differences are observed in the chemicalshifts, except for those in and near the mutated BC and FG loops. Theseresults clearly indicate that Ubi4-K retains the global fold of Fn3,despite the extensive mutations in the two loops. A few residues in theN-terminal region, which is close to the two mutated loops, also exhibitsignificant chemical differences between the two proteins. An HSQCspectrum was also recorded on a 50 μM sample of Ubi4-K in TBS. Thespectrum was similar to that collected at low pH, indicating that theglobal conformation of Ubi4 is maintained between pH 7.5 and 3.3.

The complete disclosure of all patents, patent documents andpublications cited herein are incorporated by reference as ifindividually incorporated. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

1-55. (canceled)
 56. A method for generating a protein comprising afibronectin type III domain, the method comprising removing anintegrin-binding domain from the fibronectin type III domain.
 57. Themethod of claim 56, wherein the at least one loop of the fibronectintype III domain is randomized.
 58. The method of claim 56, wherein theprotein comprises the tenth module of the fibronectin type III domain.59. (canceled)
 60. The method of claim 56, wherein the mammal is ahuman. 61-73. (canceled)
 74. A composition comprising the protein ofclaim 56.